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Cholinergic and noncholinergic tegmental pedunculopontine projection neurons in rats revealed by intracellular labeling
Abstract
Morphological features of rat pedunculopontine projection neurons were investigated in in vitro preparation by using intracellular labeling with biocytin combined with choline acetyltransferase (ChAT) immunohistochemistry. These neurons were classified into two types (Type I and II), based on their electrical membrane properties: Type I had low-threshold Ca2+ spikes, and Type II had A-current. All Type I neurons (n = 17) were ChAT immunonegative (ChAT-). Type II neurons were either ChAT immunopositive (ChAT+; n = 49) or ChAT- (n = 20). In terms of topography in the tegmental pedunculopontine nucleus (PPN), Type I neurons were dispersed throughout the extent of the nucleus, whereas Type II neurons tended to be located more in the rostral and middle sections. Both Type I and II neurons consisted of small (long axis < 20 microns), medium (20-35 microns), and large (> 35 microns) cells. The small cells were round or oval; medium cells were round, triangular, or fusiform; and the large cells were primarily fusiform in shape. In terms of the soma size, there was a difference in Type I (15-38 microns) and Type II (11-50 microns) neurons, but no significant difference was found between Type II ChAT+ and ChAT- cells. Both types of neurons had three to six primary dendrites, but the dendritic field was more prominent in Type II neurons. Most of the axons originated from one of the primary dendrites, which gave off axon collaterals, some of which projected out of the nucleus. The intrinsic collaterals were thin and branched partly within the dendritic field of the parent cell. The extrinsic collaterals were thicker and could be grouped into three categories: 1) collaterals arborizing in the substantia nigra; 2) collaterals ascending mainly toward the thalamus, pretectal, and tectal area; and 3) collaterals descending toward the mesencephalic and/or pontine reticular formation. It was noted that the collaterals of both ChAT+ and ChAT-neurons were traced into the substantia nigra. There was no significant difference in antidromic latencies between Type I (m = 1.47 msec) and Type II (m = 1.36 msec) neurons following electrical stimulation of the substantia nigra.
... Besides neurochemical groups based on neurotransmitter content [12,14,15], differences in distribution of calciumbinding proteins (rostrocaudal gradient of calbindin and calretinin; [16]) or the presence of certain neuropeptides (e.g., galanin, [5,17]) were also suggested to define subgroups of PPN neurons. NADPH diaphorase, neuronal nitric oxide synthetase (bNOS) and choline acetyltransferase were investigated as putative cholinergic PPN neuron markers [18][19][20], from which bNOS was recently proven to be a highly selective marker for cholinergic neurons [21]. Functional groups were also described in in vitro experiments, where the sorting criteria were the presence or absence of low threshold calcium spikes and transient outward potassium current (known as A-current) [19,20,22]. ...
... We grouped genetically identified cholinergic neurons with functional recordings into four functional sub-groups, according to the 'canonical' sorting criteria (n = 91) [18,19,22,37,38]. Neurons with LTS and rebound spikes (caused by calcium conductances) but without A-current and hyperpolarization-induced spike latency were considered as type I neurons. ...
... Type I neurons have low threshold spikes (due to calcium conductances), which enables them to burst firing. Type II neurons have A-current, whereas type III neurons have both properties [18,19,37,38] or none of them [22]. The latter group is sometimes referred as type IV [39]. ...
The pedunculopontine nucleus (PPN) is a part of the reticular activating system which is composed of cholinergic, glutamatergic and GABAergic neurons. Early electrophysiological studies characterized and grouped PPN neurons based on certain functional properties (i.e., the presence or absence of the A-current, spike latency, and low threshold spikes). Although other electrophysiological characteristics of these neurons were also described (as high threshold membrane potential oscillations, great differences in spontaneous firing rate and the presence or absence of the M-current), systematic assessment of these properties and correlation of them with morphological markers are still missing. In this work, we conducted electrophysiological experiments on brain slices of genetically identified cholinergic neurons in the PPN. Electrophysiological properties were compared with rostrocaudal location of the neuronal soma and selected morphometric features obtained with post hoc reconstruction. We found that functional subgroups had different proportions in the rostral and caudal subregions of the nucleus. Neurons with A-current can be divided to early-firing and late-firing neurons, where the latter type was found exclusively in the caudal subregion. Similar to this, different parameters of high threshold membrane potential oscillations also showed characteristic rostrocaudal distribution. Furthermore, based on our data, we propose that high threshold oscillations rather emerge from neuronal somata and not from the proximal dendrites. In summary, we demonstrated the existence and spatial distribution of functional subgroups of genetically identified PPN cholinergic neurons, which are in accordance with differences found in projection and in vivo functional findings of the subregions. Being aware of functional differences of PPN subregions will help the design and analysis of experiments using genetically encoded opto- and chemogenetic markers for in vivo experiments.
... Recent studies have emphasized that the dopaminergic neurons of SNc process reward-related information necessary for reinforcement learning (for review see Schultz 1998). PPTN is thought to be one of the most important input sources to SNc (Futami et al. 1995;Takakusaki et al. 1996). Accordingly, we observed an increase in the activity of PPTN neurons around reward onset (Fig. 6). ...
... PPTN receives limbic inputs from the hypothalamus, the ventral tegmental area (Semba and Fibiger 1992;Steininger et al. 1992), and the limbic cortex in monkeys (Chiba et al. 2001), all of which may be sources of the activity around reward observed in the present study. The glutamatergic and cholinergic inputs from PPTN make synaptic connections with dopaminergic neurons in SNc (Futami et al. 1995;Takakusaki et al. 1996). Electrical stimulation of PPTN induces a time-locked burst in dopaminergic neurons in the rat SNc (Lokwan et al. 1999). ...
... In a recent study of cats, reinforcement-related single-unit activity in PPTN has been demonstrated, which was preferentially observed on broadly spiking neurons presumed to be cholinergic (Dormont et al. 1998). These results suggest that cholinergic reward-related signals from PPTN may be sent to dopaminergic neurons of SNc (Takakusaki et al. 1996). ...
The cholinergic pedunculopontine tegmental nucleus (PPTN) is one of the major ascending arousal systems in the brain stem and is linked to motor, limbic, and sensory systems. Based on previous studies, we hypothesized that PPTN would be related to the integrative control of movement, reinforcement, and performance of tasks in behaving animals. To investigate how PPTN contributes to the behavioral control, we analyzed the activity of PPTN neurons during visually guided saccade tasks in three monkeys in relation to saccade preparation, execution, reward, and performance of the task. During visually guided saccades, we observed saccade-related burst (26/70) and pause neurons (19/70), indicating that a subset of PPTN neurons are related to both saccade execution and fixation. Burst neurons exhibited greater selectivity for saccade direction than pause neurons. The preferred directions for both burst and pause neurons were not aligned with either horizontal or vertical axes, nor biased strongly in either the ipsilateral or the contralateral direction. The spatial representation of the saccade-related activity of PPTN neurons is different from other brain stem saccade systems and may therefore relay saccade-related activity from different areas. Increasing discharges were observed around reward onset in a subset of neurons (22/70). These neurons responded to the freely delivered rewards within ∼140 ms. However, during the saccade task, the latencies of the responses around reward onset ranged between 100 ms before and 200 ms after the reward onset. These results suggest that the activity observed after appropriate saccade during the task may include response associated with reward. We found that the reaction time to the appearance of the fixation point (FP) was longer when the animal tended to fail in the ensuring task. This reaction time to FP appearance (RTFP) served as an index of motivation. The RTFP could be predicted by the neuronal activity of a subset of PPTN neurons (13/70) that varied their activity levels with task performance, discharging at a higher rate in successful versus error trials. A combination of responses related to saccade execution, reward delivery, and task performance was observed in PPTN neurons. We conclude from the multimodality of responses in PPTN neurons that PPTN may serve as an integrative interface between the various signals required for performing purposive behaviors.
... We also investigated the relationship between spike durations and the rhythmic firing of neurons, because previous studies reported that the neurotransmitter released by PPTN neurons might be related to their spike durations [23,24]. Spike duration was measured as the time from the negative phase to after the peak of the spike waveform [18]. ...
... Relationship between the periodic firing pattern and electrophysiological properties of neurons [23]. Anatomically, cholinergic and GABAergic neurons are distributed in different parts of the PPTN [28]. ...
... In previous studies, the neurotransmitter of recorded PPTN neurons might be related to their spike durations [23,24]; though recent study reported that distribution of spike durations from identified cholinergic and non-cholinergic neurons were not different [12]. Anatomically, cholinergic neurons are distributed to the caudal part of the PPTN, while GABAergic neurons are distributed to the rostral part [4,28]. ...
The pedunculopontine tegmental nucleus (PPTN) has been thought to be involved in the control of behavioral state. Projections to the entire thalamus and reciprocal connections with the basal ganglia nuclei suggest a potential role for the PPTN in the control of various rhythmic behaviors, including waking/sleeping and locomotion. Recently, rhythmic activity in the local field potentials was recorded from the PPTN of patients with Parkinson's disease who were treated with levodopa, suggesting that rhythmic firing is a feature of the functioning PPTN and might change with the behaving conditions even within waking. However, it remains unclear whether and how single PPTN neurons exhibit rhythmic firing patterns during various behaving conditions, including executing conditioned eye movement behaviors, seeking reward, or during resting. We previously recorded from PPTN neurons in healthy monkeys during visually guided saccade tasks and reported task-related changes in firing rate, and in this paper, we reanalyzed these data and focused on their firing patterns. A population of PPTN neurons demonstrated a regular firing pattern in that the coefficient of variation of interspike intervals was lower than what would be expected of theoretical random and irregular spike trains. Furthermore, a group of PPTN neurons exhibited a clear periodic single spike firing that changed with the context of the behavioral task. Many of these neurons exhibited a periodic firing pattern during highly active conditions, either the fixation condition during the saccade task or the free-viewing condition during the intertrial interval. We speculate that these task context-related changes in rhythmic firing of PPTN neurons might regulate the monkey's attentional and vigilance state to perform the task.
... This study supports the hypothesis that it is the cholinergic neuronal population, projecting from the PPN, which delivers some of the clinical benefits associated with PPN-DBS. Cholinergic axons project from the PPN to various basal ganglia targets, including the SNpc [21,22], the thalamus [23,24], and the striatum [25]; hence, the question remains as to which PPN cholinergic pathway is responsible for the clinical benefits associated with PPN-DBS. PPN cholinergic neurons projecting to the striatum, synapse with the nigrostriatal DAergic neurons, with studies in rodents, have demonstrated that stimulation of PPN cholinergic neurons leads to activation of nigrostriatal DAergic neurons [21,26]. ...
... Here we described experiments involving DREADD-based stimulation of neuronal cell bodies, and hence, the present study did not directly address the effects at the axonal ends. The current data contributes to guiding future work aimed at comparing output measures resulting from application of pathway-based neuromodulatory tools to PPN neurons projecting to either the SNpc of the striatum, with a large body of literature that supports the presence of such distinct PPN output pathways [6,21,22,25]. Experimental efforts to interrogate the various components of this functional network could lead to clinically exploitable substructural targets for improving PPN-DBS delivery in a clinical context. ...
The brainstem-based pedunculopontine nucleus (PPN) traditionally associates with motor function, but undergoes extensive degeneration during Parkinson’s disease (PD), which correlates with axial motor deficits. PPN-deep brain stimulation (DBS) can alleviate certain symptoms, but its mechanism(s) of action remains unknown. We previously characterized rats hemi-intranigrally injected with the proteasomal inhibitor lactacystin, as an accurate preclinical model of PD. Here we used a combination of chemogenetics with positron emission tomography imaging for in vivo interrogation of discrete neural networks in this rat model of PD. Stimulation of excitatory designer receptors exclusively activated by designer drugs expressed within PPN cholinergic neurons activated residual nigrostriatal dopaminergic neurons to produce profound motor recovery, which correlated with striatal dopamine efflux as well as restored dopamine receptor 1- and dopamine receptor 2-based medium spiny neuron activity, as was ascertained with c-Fos-based immunohistochemistry and stereological cell counts. By revealing that the improved axial-related motor functions seen in PD patients receiving PPN-DBS may be due to stimulation of remaining PPN cholinergic neurons interacting with dopaminergic ones in both the substantia nigra pars compacta and the striatum, our data strongly favor the PPN cholinergic–midbrain dopaminergic connectome as mechanism for PPN-DBS’s therapeutic effects. These findings have implications for refining PPN-DBS as a promising treatment modality available to PD patients.
... In the present study, it was not possible to determine the neurochemical nature of the recording neurons but assuming that neurons with a regular firing pattern could be the putative cholinergic type II neurons ( Takakusaki et al., 1996;Leonard and Llinás, 1994;Takakusaki et al., 1996;Kang and Kitai, 1990;Gut and Winn, 2016;Mena-Segovia and Bolam, 2017) we neither observed a modification of the non-regular neurons (putative non-cholinergic) known to receive projections from the BG output structures projections. ...
... In the present study, it was not possible to determine the neurochemical nature of the recording neurons but assuming that neurons with a regular firing pattern could be the putative cholinergic type II neurons ( Takakusaki et al., 1996;Leonard and Llinás, 1994;Takakusaki et al., 1996;Kang and Kitai, 1990;Gut and Winn, 2016;Mena-Segovia and Bolam, 2017) we neither observed a modification of the non-regular neurons (putative non-cholinergic) known to receive projections from the BG output structures projections. ...
... Finally, several studies have reported the presence of glutamate in PPN cholinergic cells in different species (Clements et al., 1991;Lavoie and Parent, 1994). In parallel, electrophysiological studies have identified substantial functional heterogeneity both within cholinergic and non-cholinergic neurons in the PPN (Steriade et al., 1990;Takakusaki et al., 1996Takakusaki et al., , 1997Mena-Segovia et al., 2008). In order to determine whether this heterogeneity might partly be due to the presence of specific cell subsets coexpressing neurotransmitters or neuromodulators, the second goal of our study was to analyze the potential coexpression of markers in the two nuclei. ...
... The ability to generate fast excitatory postsynaptic currents through the corelease of glutamate would likely confer this subset of dual cholinergic/glutamatergic cells a different electrophysiological profile to that of single cholinergic neurons. Whether any of the electrophysiological subtypes observed in the PPN (Steriade et al., 1990;Takakusaki et al., 1996Takakusaki et al., , 1997Mena-Segovia et al., 2008) corresponds to dual ChAT-and Vglut2-positive neurons remains to be analyzed. ...
The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.
... The two pathways have long been hypothesized to serve opposing functions [16,17], and recent optogenetic experiments have established opposite behavioural effects of activating each of these circuits [18][19][20][21]. The SNr and GPi contain tonically active GABAergic projection neurons that regulate motor behaviour through inhibition of regions downstream of the BG, including the thalamus, superior colliculus, dorsal raphe and brainstem motor centres [4,[22][23][24][25]. A simple model for BG regulation of target regions is that activation of dMSNs releases tonic inhibition by BG output, allowing action initiation, whereas iMSN activity increases inhibition and suppresses motor programmes, enabling an animal to stop. ...
... Indeed, there are cells in the striatum that become active at the termination of a motor sequence or at the cessation of locomotion during goal-directed behaviour [31][32][33][34][35][36][37]. As the striatum receives input from regions involved in motor planning and outcome evaluation [8,[38][39][40], and the SNr/GPi send outputs that regulate downstream motor structures [9,24,41], it would follow that information about task progress could be integrated here to cease ongoing action. In support of this, patients with Parkinson's disease (PD), which strongly disrupts striatal circuitry and function [10,11,13], have deficits in terminating ongoing movement [42,43]. ...
The ability to stop ongoing movement is fundamental to animal survival. Behavioural arrest involves the hierarchical integration of information throughout the forebrain, which ultimately leads to the coordinated inhibition and activation of specific brainstem motor centres. Recent advances have shed light on multiple regions and pathways involved in this critical behavioural process. Here, we synthesize these new findings together with previous work to build a more complete understanding of the circuit mechanisms underlying suppression of ongoing action. We focus on three specific conditions leading to behavioural arrest: goal completion, fear and startle. We outline the circuitry responsible for the production of these behaviours and discuss their dysfunction in neurological disease.
This article is part of the themed issue ‘Movement suppression: brain mechanisms for stopping and stillness’.
... neurons (Scarnati et al., 1986;Futami et al., 1995;Takakusaki et al., 1996). Electrical stimulation of the PPTg induces a burst firing of dopaminergic neurons (Lokwan et al., 1999;Floresco et al., 2003), and induces the release of dopamine in the striatum (Chapman et al., 1997;Forster and Blaha, 2003;Miller and Blaha, 2004). ...
... PD patients show a variety of motor and non-motor impairment, including PPTgrelated-symptoms, in particular, locomotor abnormalities such as shorter steps and slowness of walking, and also RAS-related deficits such as arousal and hyperactive reflexes. Anatomically, cholinergic and glutamatergic excitatory projections from the PPTg regulate activity of dopaminergic neurons in the SNc and VTA (Takakusaki et al., 1996). These observations suggest that the PPTg is related to PD (Garcia-Rill, 2015). ...
As an important component of ascending activating systems, brainstem cholinergic neurons in the pedunculopontine tegmental nucleus (PPTg) are involved in the regulation of motor control (locomotion, posture and gaze) and cognitive processes (attention, learning and memory). The PPTg is highly interconnected with several regions of the basal ganglia, and one of its key functions is to regulate and relay activity from the basal ganglia. Together, they have been implicated in the motor control system (such as voluntary movement initiation or inhibition), and modulate aspects of executive function (such as motivation). In addition to its intimate connection with the basal ganglia, projections from the PPTg to the cerebellum have been recently reported to synaptically activate the deep cerebellar nuclei. Classically, the cerebellum and basal ganglia were regarded as forming separated anatomical loops that play a distinct functional role in motor and cognitive behavioral control. Here, we suggest that the PPTg may also act as an interface device between the basal ganglia and cerebellum. As such, part of the therapeutic effect of PPTg deep brain stimulation (DBS) to relieve gait freezing and postural instability in advanced Parkinson’s disease (PD) patients might also involve modulation of the cerebellum. We review the anatomical position and role of the PPTg in the pathway of basal ganglia and cerebellum in relation to motor control, cognitive function and PD.
... The neurons found in the PPN can be divided into cholinergic, glutamatergic, and GABAergic subsets (Clements et al., 1991;Clements and Grant, 1990;Ford et al., 1995;Lavoie and Parent, 1994). The PPN subset of cholinergic neurons (CNs) has long, highly branched axons that stretch rostrally to the di-and telencephalon, as well as caudally into the brainstem (Dautan et al., 2016;Lavoie and Parent, 1994;Mena-Segovia et al., 2008;Semba and Fibiger, 1992;Takakusaki et al., 1996). These PPN CNs are more vulnerable in PD than neighboring glutamatergic and GABAergic neurons (Hirsch et al., 1987). ...
Like a handful of other neuronal types in the brain, cholinergic neurons (CNs) in the pedunculopontine nucleus (PPN) are lost during Parkinson’s disease (PD). Why this is the case is unknown. One neuronal trait implicated in PD selective neuronal vulnerability is the engagement of feed-forward stimulation of mitochondrial oxidative phosphorylation (OXPHOS) to meet high bioenergetic demand, leading to sustained oxidant stress and ultimately degeneration. The extent to which this trait is shared by PPN CNs is unresolved. To address this question, a combination of molecular and physiological approaches were used. These studies revealed that PPN CNs are autonomous pacemakers with modest spike-associated cytosolic Ca²⁺ transients. These Ca²⁺ transients were partly attributable to the opening of high-threshold Cav1.2 Ca²⁺ channels, but not Cav1.3 channels. Cav1.2 channel signaling through endoplasmic reticulum ryanodine receptors stimulated mitochondrial OXPHOS to help maintain cytosolic adenosine triphosphate (ATP) levels necessary for pacemaking. Inhibition of Cav1.2 channels led to the recruitment of ATP-sensitive K⁺ channels and the slowing of pacemaking. A ‘side-effect’ of Cav1.2 channel-mediated stimulation of mitochondria was increased oxidant stress. Thus, PPN CNs have a distinctive physiological phenotype that shares some, but not all, of the features of other neurons that are selectively vulnerable in PD.
... 运动过程中, 步态控制的自动化过程由从脑干到 脊髓的下行神经环路介导, 该环路是动物稳健步行和 运动的关键 [7] . 正常情况下, 参与步态调控的脑网络包 [16,17] . MLR主要包含两个核团: 脚桥核 (pedunculopontine nucleus, PPN)和楔形核(cuneiform 图 1 正常步态控制神经网络 Figure 1 Neural networks of normal gait control nucleus, CnF) [14,18] , 且MLR由谷氨酸能、γ-氨基丁酸 (gamma-aminobutyric acid, GABA)能和胆碱能三类神 经元组成, 分别行使着异质性功能 [17,19] . ...
... In the rat and cat, cholinergic neurons of the PPN innervate cells across a large portion of the hindbrain reticular formation. Anterograde tracing experiments revealed that the pars compacta of the cat PPN sends descending projections that reach the pontine and bulbar reticular formation (Edley and Graybiel, 1983;Skinner et al., 1990;Inglis and Winn, 1995;Karachi et al., 2010;Martinez-Gonzalez et al., 2011;Mazzone et al., 2012) and 47% of descending axons to the pontine reticular formation are from PPN cholinergic cells (Takakusaki et al., 1996). Projections to the spinal cord have been reported in the rat (Rye et al., 1988;Spann and Grofova, 1989), but not in the cat (Edley and Graybiel, 1983). ...
Over the last 60 years, the basic neural circuitry responsible for the supraspinal control of locomotion has progressively been uncovered. Initially, significant progress was made in identifying the different supraspinal structures controlling locomotion in mammals as well as some of the underlying mechanisms. It became clear, however, that the complexity of the mammalian central nervous system (CNS) prevented researchers from characterizing the detailed cellular mechanisms involved and that animal models with a simpler nervous system were needed. Basal vertebrate species such as lampreys, xenopus embryos, and zebrafish became models of choice. More recently, optogenetic approaches have considerably revived interest in mammalian models. The mesencephalic locomotor region (MLR) is an important brainstem region known to control locomotion in all vertebrate species examined to date. It controls locomotion through intermediary cells in the hindbrain, the reticulospinal neurons (RSNs). The MLR comprises populations of cholinergic and glutamatergic neurons and their specific contribution to the control of locomotion is not fully resolved yet. Moreover, the downward projections from the MLR to RSNs is still not fully understood. Reporting on discoveries made in different animal models, this review article focuses on the MLR, its projections to RSNs, and the contribution of these neural elements to the control of locomotion. Excellent and detailed reviews on the brainstem control of locomotion have been recently published with emphasis on mammalian species. The present review article focuses on findings made in basal vertebrates such as the lamprey, to help direct new research in mammals, including humans.
... Anatomical evidence for differential subcellular localization of ACh and GABA presynaptic release sites along the somatodendritic extent of SN DA neurons SN DA neurons are innervated by longrange cholinergic projections from the laterodorsal tegmental and pedunculopontine tegmental nuclei (PPN) (Clarke et al., 1987;Cornwall et al., 1990;Futami et al., 1995;Takakusaki et al., 1996;Lokwan et al., 1999;Xiao et al., 2016;Estakhr et al., 2017;Li and Spitzer, 2020). To further validate the differential nature of ACh and GABA synaptic release from cholinergic axons onto medial SN DA neurons, we used ChATcre(neo-del) 1/À :: Ai9-tdTomato 1/À mice that show selective labeling of cholinergic neurons and their axonal processes (Nasirova et al., 2020). ...
We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: co-transmitted acetylcholine (ACh)/GABA, GABA only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA co-transmission. In response to repeated stimulation the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA co-transmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by co-transmitted ACh/GABA was more effective in eliciting an action potential compared to current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels; thus, establishing that activity of multiple classes of ion channels integrate to shape neuronal excitability.SIGNIFICANCE STATEMENT:Our lab has previously discovered a population of substantia nigra (SN) dopaminegic neurons (DA) that receive co-transmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of SN DA neurons. We determined spatially clustered GABA only inputs on the lateral dendrites while co-transmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in co-transmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T-type and L-type voltage-gated calcium channels.
... Therefore, we suggest that glutamate increases the cardiovascular responses by activation of these neurons. It has been shown that PPT has two cholinergic and noncholinergic projections to several brain areas including areas involved in cardiovascular regulation (12,34,35). It has been proposed that the non-cholinergic projections of PPT are glutamatergic or GABAergic. ...
Objectives:
In the present study, the cardiovascular effects of glutamate NMDA receptor of the pedunculopontine tegmental nucleus (PPT) in normotensive and hydralazine (HLZ) hypotensive rats were evaluated.
Materials and methods:
In the normotensive condition, MK-801(1 nmol; an NMDA receptor antagonist) and L-glutamate (L-Glu, 50 nmol an agonist) alone and together were microinjected into the nucleus using a stereotaxic device. In hypotensive condition, 2 min after induction of hypotension by HLZ (10 mg/kg, intravenous), drugs, same as in normotensive condition, were microinjected into the PPT. Recorded mean arterial pressure (MAP), systolic blood pressure (SBP), and heart rate (HR) were recorded throughout the experiment by a Power lab apparatus that was connected to a catheter inserted into the femoral arty. The cardiovascular changes (Δ) induced by microinjection drugs were computed and statistically analyzed.
Results:
In the normotensive condition, L-Glu significantly increased ΔMAP and ΔSBP (P<0.001) and decreased ΔHR (P<0.01) compared with the control. MK-801 alone significantly increased HR (P<0.05) while co-injected with L-Glu + MK-801 it significantly attenuated the L-Glu effect on ΔMAP and ΔSBP but augmented ΔHR (P<0.01). In the hydralazine hypotension condition, L-Glu significantly improved hypotension (P<0.01) and deteriorated bradycardia induced by HLZ (P<0.05). MK-801 alone did not significantly affect ΔMAP, ΔSBP, and ΔHR but when co-injected with L-Glu (L-Glu + MK-801) it could significantly attenuate the cardiovascular effect of L-Glu in the PPT.
Conclusion:
We found that activation of NMDA receptors of the glutamatergic system in the PPT evoked blood pressure and inhibited HR in both normotensive and hypotensive conditions in rats.
... Importantly, it is glutamatergic mesencephalic reticular formation neurons, including regions of the CnF, SubCnF, and PPT, that are activated during treadmill locomotion and which may code for locomotor speed (Roseberry et al., 2016;Caggiano et al., 2018). Cholinergic neurons, in contrast are characterized by repetitive, slow firing (Takakusaki et al., 1996). In non-human primates, rhythmically active cells are preferentially located in more dorsal CnF and SubCnF locations than tonically activated ones (Goetz et al., 2016). ...
The distribution of locomotor-activated neurons in the brainstem of the cat was studied by c-Fos immunohistochemistry in combination with antibody-based cellular phenotyping following electrical stimulation of the mesencephalic locomotor region (MLR) – the anatomical constituents of which remain debated today, primarily between the cuneiform (CnF) and the pedunculopontine tegmental nuclei (PPT). Effective MLR sites were co-extensive with the CnF nucleus. Animals subject to the locomotor task showed abundant Fos labeling in the CnF, parabrachial nuclei of the subcuneiform region, periaqueductal gray, locus ceruleus (LC)/subceruleus (SubC), Kölliker–Fuse, magnocellular and lateral tegmental fields, raphe, and the parapyramidal region. Labeled neurons were more abundant on the side of stimulation. In some animals, Fos-labeled cells were also observed in the ventral tegmental area, medial and intermediate vestibular nuclei, dorsal motor nucleus of the vagus, n. tractus solitarii, and retrofacial nucleus in the ventrolateral medulla. Many neurons in the reticular formation were innervated by serotonergic fibers. Numerous locomotor-activated neurons in the parabrachial nuclei and LC/SubC/Kölliker–Fuse were noradrenergic. Few cholinergic neurons within the PPT stained for Fos. In the medulla, serotonergic neurons within the parapyramidal region and the nucleus raphe magnus were positive for Fos. Control animals, not subject to locomotion, showed few Fos-labeled neurons in these areas. The current study provides positive evidence for a role for the CnF in the initiation of locomotion while providing little evidence for the participation of the PPT. The results also show that MLR-evoked locomotion involves the parallel activation of reticular and monoaminergic neurons in the pons/medulla, and provides the anatomical and functional basis for spinal monoamine release during evoked locomotion. Lastly, the results indicate that vestibular, cardiovascular, and respiratory centers are centrally activated during MLR-evoked locomotion. Altogether, the results show a complex pattern of neuromodulatory influences of brainstem neurons by electrical activation of the MLR.
... Neurons in MLR are excitatory. The pedunculopontine nucleus (PPN) is located in the ventrolateral portion of caudal MLR and is composed of neurons comprising glutamate and acetylcholine (ACh) (Clements & Grant, 1990;Lavoie & Parent, 1994;Takakusaki et al., 1996;Mena-Segovia et al., 2008;Takakusaki et al., 2016). Previous studies have suggested that cholinergic neurons are important in maintaining the rhythm of locomotion and postural muscle tone (Bohnen & Albin, 2011;Takakusaki et al., 2011). ...
Background: Mammalian locomotor behaviour called fictive locomotion can be elicited in an isolated spinal cord in the absence of higher brain center or sensory input. This relatively simple behaviour is produced by the motoneuronal rhythmic activity which is under the control of spinal neuronal networks called central pattern generators (CPGs). Disturbance of this rhythmic motor output can occur following spinal cord injury (SCI). This elementary isolated spinal cord model gives us an opportunity to study the basic physiology of locomotion during control conditions, the pathological processes following lesion (which can be induced chemically), and eventually the application of therapeutic approaches curbing injury.
Objectives: Multiple aspects of spinal functions can be demonstrated by stimulating or/and blocking specific inputs and measuring the outputs using electrophysiological, immunohistochemical and calcium imaging tools. Using isolated neonatal rat spinal cords and organotypic spinal slices as SCI models, the basic mechanisms (such as dysmetabolic state or excitotoxicity) which can develop during the early phase of the lesion were addressed and studied. The injury was evoked chemically by applying either pathological medium (to mimic dysmetabolic/hypoxic conditions) or kainate (to produce excitotoxicity that completely abolishes fictive locomotion and network synaptic transmission) for 1 h. Fictive locomotion was examined stimulating the lumbar dorsal root and recording from the ipsilateral and ipsi-segmental ventral roots. Other network parameters were also studied such as synaptic transmission and rhythmicity. Various therapeutic drugs such as methylprednisolone sodium succinate (MPSS), propofol, nicotine and celastrol were used during or after the injury (to produce neuroprotection) and network properties were characterized during the treatment and after 24 h as well. Subsequently, the structural properties were monitored using different biomarkers (isolated spinal cord sectioned slices) and calcium imaging (here organotypic spinal slices were used).
Results and conclusions: We found that dose-dependent application of MPSS produced modest recovery of white matter damage evoked by pathological medium resulting in the emergence of sluggish chemically induced fictive locomotor patterns. However, it could not prevent damage (to gray matter) evoked by the excitotoxic agent kainate. Therefore, to provide better neuroprotection to gray matter, we tested the widely used intravenous anaesthetic propofol. This drug has shown comparatively good protection to spinal neurons and motoneurons in the gray matter. As it is an anesthetic it acted by depressing the functional network characteristics by lowering the N-methyl-D-aspartate (NMDA) and potentiating the γ aminobutyric acid (GABAA) mediated receptor responses.
The next issue we addressed was to study the neuroprotective roles of nicotinic acetylcholine receptors (nAChRs) by using the receptor agonist nicotine. Recent studies have shown that nicotine could provide good neuroprotection to the rat brainstem. To further investigate its effect on the spinal cord, we applied nicotine at the same concentration used in previous studies in the brainstem: such a concentration was toxic to spinal ventral motoneurons. Therefore the correct dose of nicotine was optimized and was found to be ten times lower. Thus, satisfactory protective effects to spinal neurons and motoneurons and the fictive locomotor patterns were observed. These neuroprotective effects were replicated with calcium imaging by using organotypic spinal slice cultures. The mechanism of protection predominantly involved α4β2 and less α7 nAChRs.
In addition, the subsequent goal of our study was to explore whether the motoneuron survival after excitotoxicity relies on cell expression of heat shock protein 70 (HSP70) or some other mechanisms. To test this hypothesis we used a bioactive drug, celastrol which induces the expression of HSP70. Prior application of the drug followed by kainate preserved network polysynaptic transmission and fictive locomotion, however, it could not reverse the depression of monosynaptic reflex responses. In vivo studies are necessary in the future to further investigate the long-term neuroprotective role of these drugs.
... Another way could be to record neurons in awake animals. While the neurons of the PPN have already been studied based on their electrophysiological properties (Takakusaki et al., 1996;Roš et al., 2010;Petzold et al., 2015), the studies focused on the PPN at large and not on its dorsal part or its anatomical boundary with the CnF. Recording in multiple sites across the brainstem in awake animals or in patients implanted with DBS electrodes would help resolve this anatomical conundrum. ...
... The efferent connectome of the PPN also is diffuse. The longest ranging PPN projections arise from the cholinergic neurons, which as a population, send both ascending projections to the thalamus, basal ganglia, and limbic structures and descending projections to other brainstem nuclei (Dautan et al., 2016;Dautan et al., 2014;Lavoie and Parent, 1994c;Mena-Segovia et al., 2008;Semba and Fibiger, 1992;Takakusaki et al., 1996) (Fig. 1). In contrast, the GABAergic and glutamatergic neurons appear to largely target neighboring areas within the midbrain and brainstem (Bevan and Bolam, 1995;Ford et al., 1995;Mena-Segovia et al., 2008;Ros et al., 2010) (Fig. 1). ...
In the last decade, scientific and clinical interest in the pedunculopontine nucleus (PPN) has grown dramatically. This growth is largely a consequence of experimental work demonstrating its connection to the control of gait and of clinical work implicating PPN pathology in levodopa-insensitive gait symptoms of Parkinson's disease (PD). In addition, the development of optogenetic and chemogenetic approaches has made experimental analysis of PPN circuitry and function more tractable. In this brief review, recent findings in the field linking PPN to the basal ganglia and PD are summarized; in addition, an attempt is made to identify key gaps in our understanding and challenges this field faces in moving forward.
... Substantia nigra pars reticulata receive glutamatergic afferents from the cortex (Naito & Kita, 1994), the pedunculopontine nucleus (Takakusaki, Shiroyama, Yamamoto, & Kitai, 1996), the zona incerta (Heise & Mitrofanis, 2004), and from the subthalamic nucleus (Parent & Hazrati, 1995); thus, modulation by CB2r can occur in any of these. However, it has been demonstrated that lesions of the subthalamus induced with kainic acid, as employed in this study, reduces 80% of glutamate in the substantia nigra (Rosales et al., 1997), indicating that this is the major source of K + -induced [ 3 H]-Glutamate release and, consequently, the site of modulation by CB2r. ...
Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appears is expressed in the subthalamic nucleus. Here we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose‐dependent inhibited K⁺‐induced [³H]‐Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE‐907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [³H]‐Glutamate release was PTX‐ and gallein‐sensitive, suggesting a Giβγ‐mediated effect. P/Q Ca²⁺‐type channel blocker, ω‐Agatoxin‐TK, also inhibited the [³H]‐Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the βγ subunit effect is exerted on Ca²⁺ channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [³H]‐Glutamate release in subthalamo‐nigral terminals by P/Q‐channels modulation through the Giβγ subunit and suggested their participation in motor behavior.
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... In a recent publication (138), we demonstrated in vitro that reducing the axonal arbor size of SNc DA neurons to a size more similar to that of VTA DA neurons using the axonal guidance factor Semaphorin 7A, was sufficient to greatly reduce basal OXPHOS and reduce their vulnerability to toxins including MPP+ and rotenone. Although as previously discussed, the extent of neuronal loss is still unclear for many neuronal populations, it does seem likely that most neuronal nuclei affected in PD include neurons that are relatively few in number, but all possess long and profuse unmyelinated axonal arbors and a large number of axonal terminals (171)(172)(173)(174)(175)(176). However, comparative data evaluating axonal arbor size amongst these populations and in populations of neurons that do not degenerate in PD is presently lacking. ...
Significant advances have been made uncovering the factors that render neurons vulnerable in Parkinson's disease (PD). However, the critical pathogenic events leading to cell loss remain poorly understood, complicating the development of disease-modifying interventions. Given that the cardinal motor symptoms and pathology of PD involve the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc), a majority of the work in the PD field has focused on this specific neuronal population. PD however, is not a disease of DA neurons exclusively: pathology, most notably in the form of Lewy bodies and neurites, has been reported in multiple regions of the central and peripheral nervous system, including for example the locus coeruleus, the dorsal raphe nucleus and the dorsal motor nucleus of the vagus. Cell and/or terminal loss of these additional nuclei is likely to contribute to some of the other symptoms of PD and, most notably to the non-motor features. However, exactly which regions show actual, well-documented, cell loss is presently unclear. In this review we will first examine the strength of the evidence describing the regions of cell loss in idiopathic PD, as well as the order in which this loss occurs. Secondly, we will discuss the neurochemical, morphological and physiological characteristics that render SNc DA neurons vulnerable, and will examine the evidence for these characteristics being shared across PD-affected neuronal populations. The insights raised by focusing on the underpinnings of the selective vulnerability of neurons in PD might be helpful to facilitate the development of new disease-modifying strategies and improve animal models of the disease.
... Functional studies performed on anesthetized animals show that, after electrical stimulation of the SN, inhibitory postsynaptic potentials were recorded in PPTg, both in rats (Granata and Kitai 1991) and cats (Noda and Oka 1986). Similar conclusions were also reached in other studies using brain slices (Kang and Kitai 1990;Takakusaki et al. 1996). ...
The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.
... The PPN contains cholinergic, glutamatergic, and GABAergic neurons [3]. The cholinergic output has a net excitatory effect on LC and RN neurons [4,5]. Importantly, one of the targets of PPN non-cholinergic neurons is the VTA [6]. ...
... 120 Cholinergic PPN neurons project to the non-specific thalamocortical system, 121-123 basal ganglia nuclei including DA neurons in the SNc and PMRF. [124][125][126] On the other hand, cholinergic BF projections to the cerebral cortex are necessary for attentional performance and cognitive processing. 127 Therefore, disturbances in attention, sensori-motor integration and cognitive processing in PD can be largely attributed to the damage of the cholinergic systems. ...
Here we argue functional neuroanatomy for posture- gait control. Multi-sensory information such as somatosensory, visual and vestibular sensation act on various areas of the brain so that adaptable posture- gait control can be achieved. Automatic process of gait, which is steady-state stepping movements associating with postural reflexes including headeye coordination accompanied by appropriate alignment of body segments and optimal level of postural muscle tone, is mediated by the descending pathways from the brainstem to the spinal cord. Particularly, reticulospinal pathways arising from the lateral part of the mesopontine tegmentum and spinal locomotor network contribute to this process. On the other hand, walking in unfamiliar circumstance requires cognitive process of postural control, which depends on knowledges of self-body, such as body schema and body motion in space. The cognitive information is produced at the temporoparietal association cortex, and is fundamental to sustention of vertical posture and construction of motor programs. The programs in the motor cortical areas run to execute anticipatory postural adjustment that is optimal for achievement of goal-directed movements. The basal ganglia and cerebellum may affect both the automatic and cognitive processes of posturegait control through reciprocal connections with the brainstem and cerebral cortex, respectively. Consequently, impairments in cognitive function by damages in the cerebral cortex, basal ganglia and cerebellum may disturb posture-gait control, resulting in falling.
... Previous work has shown that the MLR has robust descending projections to the gigantocellular nucleus (Martinez-Gonzalez et al., 2014;Mitani et al., 1988;Rye et al., 1988), also referred to as the ventromedial medulla (Sherman et al., 2015;Skinner et al., 1990). In addition, MLR axons and terminals have been found in the pontine reticular formation (Takakusaki et al., 1996) and nucleus pontine oralis (Garcia-Rill et al., 2001). Collectively, these nuclei form the origin of reticulospinal tracts that project into the spinal cord and mediate various aspects of posture and movement. ...
The basal ganglia (BG) are critical for adaptive motor control, but the circuit principles underlying their pathway-specific modulation of target regions are not well understood. Here, we dissect the mechanisms underlying BG direct and indirect pathway-mediated control of the mesencephalic locomotor region (MLR), a brainstem target of BG that is critical for locomotion. We optogenetically dissect the locomotor function of the three neurochemically distinct cell types within the MLR: glutamatergic, GABAergic, and cholinergic neurons. We find that the glutamatergic subpopulation encodes locomotor state and speed, is necessary and sufficient for locomotion, and is selectively innervated by BG. We further show activation and suppression, respectively, of MLR glutamatergic neurons by direct and indirect pathways, which is required for bidirectional control of locomotion by BG circuits. These findings provide a fundamental understanding of how BG can initiate or suppress a motor program through cell-type-specific regulation of neurons linked to specific actions.
... Stimulation of the pedunculopontine nucleus in vivo produces mostly excitation of dopaminergic neurons at short latencies ranging from 3 to 5 ms (Scarnati et al. 1984), consistent with the conduction time of cholinergic neurons from the pedunculopontine nucleus to the substantia nigra (Futami et al. 1995;Takakusaki et al. 1996). The EPSP that underlies the excitation seen extracellularly in vivo is composed of both nicotinic and pirenzepinesensitive muscarinic components (Futami et al. 1995). ...
We dedicate this chapter to the memory of Dr. Stephen J. Young, mentor, colleague and friend. For decades Steve contributed tirelessly and selflessly to the advancement of the science of countless students, colleagues and scientists around the world. His presence is sorely missed.
... This is important because the basal activity of VTA neurons can be functionally associated to individual vulnerability to psychostimulant abuse (Marinelli and White, 2000). The PPN nucleus contains cholinergic and noncholinergic neurons that have an excitatory effect on LC and RN neurons (Egan and North, 1986; Takakusaki et al., 1996). It is important to note that one of the targets of PPN C H A P T E R ...
Interactions between the pedunculopontine (PPN) and ventral tegmental area (VTA) have been described as key brain circuitry that mediates psychostimulant-mediated reward. Intranuclear PPN/laterodorsal tegmental injections of cholinergic receptor agonists or antagonists have helped clarify the role of these nuclei on psychostimulant-induced locomotion. Also, nicotine has been shown to have an inhibitory effect on the PPN, at least initially reducing arousal. PPN activation has been involved in the animal's voluntary search for psychostimulants. On the other hand, CNS depressants like ethanol might reduce arousal by a mechanism involving the direct inhibition of the PPN. In conclusion, neural activity in the PPN is key not only to maintaining arousal but also to affecting the level of psychostimulant and depressant administration.
... Therefore, a consistent correlation between the proposed classification system and the neurotransmitter phenotype of recorded PPTg neurons displaying Type I, II, III or IV properties remains to be confirmed, particularly in in vivo preparations. Moreover, the classification scheme only allows for distinguishing between cholinergic and non-cholinergic neurons in general, with neurophysiological profiles correlating to a single Type, which were revealed to consist of "mixed" populations, containing both cholinergic and non-cholinergic neurons [37,42]. A study by Zhang and colleagues [43] found that the majority of presumed cholinergic and non-cholinergic neurons in the unlesioned rat PPTg show a regular firing pattern in normal rats, while in rats that had received an SNc lesion of 6-OHDA, an increased percentage of presumed non-cholinergic neurons exhibited an irregular firing pattern, while the firing pattern of presumed cholinergic neurons remained similar to those recorded in normal rats. ...
Background:
Patients with advanced Parkinson's disease (PD) often present with axial symptoms, including postural- and gait difficulties that respond poorly to dopaminergic agents. Although deep brain stimulation (DBS) of a highly heterogeneous brain structure, the pedunculopontine nucleus (PPN), improves such symptoms, the underlying neuronal substrate responsible for the clinical benefits remains largely unknown, thus hampering optimization of DBS interventions. Choline acetyltransferase (ChAT)::Cre(+) transgenic rats were sham-lesioned or rendered parkinsonian through intranigral, unihemispheric stereotaxic administration of the ubiquitin-proteasomal system inhibitor, lactacystin, combined with designer receptors exclusively activated by designer drugs (DREADD), to activate the cholinergic neurons of the nucleus tegmenti pedunculopontine (PPTg), the rat equivalent of the human PPN. We have previously shown that the lactacystin rat model accurately reflects aspects of PD, including a partial loss of PPTg cholinergic neurons, similar to what is seen in the post-mortem brains of advanced PD patients.
Results:
In this manuscript, we show that transient activation of the remaining PPTg cholinergic neurons in the lactacystin rat model of PD, via peripheral administration of the cognate DREADD ligand, clozapine-N-oxide (CNO), dramatically improved motor symptoms, as was assessed by behavioral tests that measured postural instability, gait, sensorimotor integration, forelimb akinesia and general motor activity. In vivo electrophysiological recordings revealed increased spiking activity of PPTg putative cholinergic neurons during CNO-induced activation. c-Fos expression in DREADD overexpressed ChAT-immunopositive (ChAT+) neurons of the PPTg was also increased by CNO administration, consistent with upregulated neuronal activation in this defined neuronal population.
Conclusions:
Overall, these findings provide evidence that functional modulation of PPN cholinergic neurons alleviates parkinsonian motor symptoms.
... (i) There is a genuine difference in the projection patterns of the slightly more rostral vlPRF anticonvulsant zone to the thalamus and pretectum, compared with ventrolateral tegmental region studied by Herbert et al. (1997), the rostal vlPRF providing a more significant input. (ii) There are significant populations of neurons in afferent structures, such as the superior colliculus (Chevalier & Deniau, 1984;Redgrave et al., 1986;Bickford & Hall, 1989), the pedunculopontine nucleus (Takakusaki et al., 1996) and periaqueductal grey (Reichling & Basbaum, 1991), that have collateral efferent projections with both ascending and descending components. In these cases, one axonal branch descends to innervate hindbrain structures, possibly including the vlPRF, while an ascending collateral projects forwards to terminate strongly in the intralaminar thalamus and pretectum. ...
... In the VTA to NAc circuit, DA burst firing and phasic DA release is mediated by N-methyl-D-aspartate receptor (NMDAR) and acetylcholine receptor (AChR) mechanisms Blaha, 2000, 2003;Grace et al., 2007;Sombers et al., 2009;Wickham et al., 2013) and recent evidence points toward a role for VTA NMDARs and AChRs in cue-dependent behavior. In particular, the mesopontine tegmentum (MPT), which contains the laterodorsal tegmentum (LDTg) and pedunculopontine tegmentum (PPTg), sends both cholinergic and glutamatergic projections to the VTA (Clements et al., 1991;Honda and Semba, 1995;Oakman et al., 1995;Takakusaki et al., 1996). PPTg inactivation has been shown to impair stimulus-reward learning, conditioned reinforcement (Inglis et al., 2000) and also impairs the ability of VTA DA neurons to burst fire in the presence of reward-predictive cues (Pan et al., 2005). ...
Stimuli paired with rewards acquire reinforcing properties to promote reward-seeking behavior. Previous work supports the role of ventral tegmental area (VTA) nicotinic acetylcholine receptors (nAChRs) in mediating conditioned reinforcement elicited by drug-associated cues. However, it is not known whether these cholinergic mechanisms are specific to drug-associated cues or whether VTA cholinergic mechanisms also underlie the ability of cues paired with natural rewards to act as conditioned reinforcers. Burst firing of VTA dopamine (DA) neurons and the subsequent phasic DA release in the nucleus accumbens (NAc) plays an important role in cue-mediated behavior and in the ability of cues to acquire reinforcing properties. In the VTA, both AChRs and N-methyl-d-aspartate receptors (NMDARs) regulate DA burst firing and phasic DA release. Here, we tested the role of VTA nAChRs, muscarinic AChRs (mAChRs), and NMDARs in the conditioned reinforcement elicited by a food-associated, natural reward cue. Subjects received 10 consecutive days of Pavlovian conditioning training where lever extension served as a predictive cue for food availability. On day 11, rats received bilateral VTA infusion of saline, AP-5 (0.1 or 1 μg), mecamylamine (MEC: 3 or 30 μg) or scopolamine (SCOP: 3 or 66.7 μg) immediately prior to the conditioned reinforcement test. During the test, nosepoking into the active (conditioned reinforced, CR) noseport produced a lever cue while nosepoking on the inactive (non-conditioned reinforced, NCR) noseport had no consequence. AP-5 robustly attenuated conditioned reinforcement and blocked discrimination between CR and NCR noseports at the 1-μg dose. MEC infusion decreased responding for both CR and NCR while 66.7-μg SCOP disrupted the subject’s ability to discriminate between CR and NCR. Together, our data suggest that VTA NMDARs and mAChRs, but not nAChRs, play a role in the ability of natural reward-associated cues to act as conditioned reinforcers.
... Les sources de glutamate de la SNr chez le rat proviennent des afférences corticales (Naito and Kita, 1994), de celles du PPN (Takakusaki et al., 1996) mais principalement de celles du NST (Robledo and Feger, 1990) dont les fibres arborisent toute la SNr en formant des synapses sur les dendrites proximales et distales (Parent and Hazrati, 1995a (Gotz et al., 1997;Chatha et al., 2000). ...
Basal ganglia, a set of interconnected nuclei, are implicated in the elaboration, control and memorization of cognitive-motor behaviors. One of the main output structure of this network, the substantia nigra pars reticulata (SNr), integrates and conveys neuronal information to cortical areas via a thalamic relay. However, this transmission requires an accurate regulation of the SNr neuronal activity since this structure inhibits its targets due to its spontaneous GABAergic activity. Among the different actors of this regulation, glutamate and GABA provide a tight balance between excitation and inhibition of the SNr neuronal activity. Several studies have explored the different mechanisms involved in this regulation but paradoxically, none concerned the astrocyte functions. In this work, our aim was to study astrocyte-neuron relations in order to define a potential astrocyte implication in the regulation of the neuronal activity in the SNr. We studied calcium and electrical activities of astrocytes and neurons using calcium imaging and patch-clamp techniques in parasagittal rat brain slices, conserving subthalamo- and pallido-nigral projections. We showed that astrocytes in the SNr displayed spontaneous calcium activities, both dependent and independent of glutamatergic and GABAergic tonic neuronal transmissions. Moreover, we showed that astrocytes calcium activities were regulated by the subthalamic nucleus high frequency stimulation. Our results revealed that, in turn, astrocytes calcium activities were involved in the regulation of the neuronal firing rate. Finally, we showed that astrocyte glutamatergic, and maybe GABAergic, reuptake was involved in the regulation of the neuronal firing rate. To conclude, this study revealed a bidirectional communication between astrocytes and neurons in the SNr. This communication seems to be important in the regulation of the activity in this structure.
... Chez l'homme, l'étude indirecte, par imagerie cérébrale, de la connectivité du PPN montre les mêmes projections que chez le primate non-humain, incluant les aires motrices corticales, les ganglions de la base, le thalamus, le cervelet et la moelle épinière Muthusamy et al., 2007), autrement dit des connexions avec les structures clefs du contrôle moteur. Le PPN reçoit aussi des afférences gabaergiques et glutamatergiques en provenance respectivement de la substance noire réticulée et du NST , et il entretient des relations avec le système dopaminergique grâce à ses efférences vers la substance noire compacte (Charara et al., 1996 ;Takakusaki et al., 1996). Chez le singe, le PPN et le noyau cunéiforme reçoivent une projection dopaminergique, fortement réduite après lésion au MPTP (Rolland et al., 2009). ...
Gait disorders in the healthy elderly are a major public health concern. We believed understanding Parkinson Disease (PD) typical gait disorders would bring some insights into the mechanisms of gait disorders in the elderly. In PD, their response to levodopa and to subthalamic nucleus stimulation is often heterogeneous, suggesting they are controlled at different levels of the CNS. In particular, there is convergent evidence of early alterations in cholinergic neurotransmission responsible for levodopa-resistant gait disorders in PD. An example comes from the observed link between executive dysfunction and the presence of levodopa-resistant freezing. Moreover, the relative preservation of executive functions in some patients suggests that other mechanisms may be involved in the development of levodopa-resistant freezing. The pedunculopontine nucleus area (PPNa) is a likely candidate. We report a beneficial effect of PPNa stimulation on freezing and falls related to freezing in some patients. However, our results are disappointing compared to the high levels of expectation raised by previous open label studies. Further controlled studies are needed to determine whether optimization of patient selection, targeting and setting of stimulation parameters might improve the outcome to a point that could transform this experimental approach to a treatment with a reasonable risk–benefit ratio.
... STN and PPN axons form asymmetric synapses on medium-sized and small SNc dendrites. The PPN boutons tend to form synapses onto larger diameter dendrites than the STN boutons (Hammond et al., 1978;Jackson and Crossman, 1983;Scarnati et al., 1984;Rye et al., 1987;Rinvik and Ottersen, 1993;Futami et al., 1995;Takakusaki et al., 1996). ...
The spontaneous activity pattern of adult dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) results from interactions between intrinsic membrane conductances and afferent inputs. In adult SNc DA neurons, low-frequency tonic background activity is generated by intrinsic pacemaker mechanisms, whereas burst generation depends on intact synaptic inputs in particular the glutamatergic ones. Tonic DA release in the striatum during pacemaking is required to maintain motor activity, and burst firing evokes phasic DA release, necessary for cue-dependent learning tasks. However, it is still unknown how the firing properties of SNc DA neurons mature during postnatal development before reaching the adult state. We studied the postnatal developmental profile of spontaneous and evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in SNc DA neurons in brain slices from immature (postnatal days P4-10) and young adult (P30-50) tyrosine hydroxylase (TH)-GFP mice. We found that somato-dendritic fields of SNc DA neurons are already mature at P4-10. In contrast, spontaneous glutamatergic EPSCs show a developmental sequence. Spontaneous NMDA EPSCs in particular are larger and more frequent in immature SNc DA neurons than in young adult ones and have a bursty pattern. They are mediated by GluN2B and GluN2D subunit-containing NMDA receptors. The latter generate long-lasting, DQP1105-sensitive, spontaneous EPSCs, which are transiently recorded during this early period. Due to high NMDA activity, immature SNc DA neurons generate large and long lasting NMDA receptor-dependent (APV-sensitive) bursts in response to the stimulation of the subthalamic nucleus. We conclude that the transient high NMDA activity allows calcium influx into the dendrites of developing SNc DA neurons.
... The PPNc/MEA provides substantial cholinergic/glutamatergic innervation of the SNc (Parent and Hazrati, 1995;Takakusaki, 1996), and reciprocates the input it receives from the STN with a substantial, excitatory projection, as well (Woolf and Butcher, 1986;Canteras, 1990;Inglis and Winn, 1995). This would seem to leave the PPNc/MEA in an ideal position to support cellular activity in the STN of decerebrate rat pups, and to coordinate the behavioral response. ...
... PPN projections to the SNc in non-human primates and rats have been shown to consist of both cholinergic and glutamatergic neurons. Only a minor contingent of PPN cholinergic neurons project to SNr (Lavoie and Parent, 1994a;Charara et al., 1996;Takakusaki et al., 1996). The STN receives bilateral projections from the PPN in the rat (Woolf and Butcher, 1986), cat (Edley and Graybiel, 1983) and monkey (Lavoie and Parent, 1994b). ...
Gait and balance abnormalities develop commonly in Parkinson's disease and are among the motor symptoms most disabling and refractory to dopaminergic or other treatments, including deep brain stimulation. Efforts to develop effective therapies are challenged by limited understanding of these complex disorders. There is a major need for novel and appropriately targeted research to expedite progress in this area. The Scientific Issues Committee of the International Parkinson and Movement Disorder Society has charged a panel of experts in the field to consider the current knowledge gaps and determine the research routes with highest potential to generate groundbreaking data. © 2021 International Parkinson and Movement Disorder Society.
The basal ganglia are several synaptically interconnected subcortical structures that play important roles in regulating various aspects of psychomotor behaviors, and are central to the pathophysiology of common human movement disorders such as Parkinson’s and Huntington’s diseases (PD/HD). These structures classically include: 1) the striatum, which comprises the caudate nucleus (CD), putamen (PUT), and nucleus accumbens (Acc); 2) the globus pallidus, which includes the external (GPe; globus pallidus in nonprimates) and internal (GPi; entopeduncular nucleus [EPN] in nonprimates) segments; 3) the subthalamic nucleus (STN); and 4) the substantia nigra, which comprises the pars compacta (SNc) and pars reticulata (SNr) (Fig. 1).
The interest in the pedunculopontine tegmental nucleus (PPTg), a structure located in the brainstem at the level of the pontomesencephalic junction, has greatly increased in recent years because it is involved in the regulation of physiological functions that fail in Parkinson's disease and because it is a promising target for deep brain stimulation in movement disorders. The PPTg is highly interconnected with the main basal ganglia nuclei and relays basal ganglia activity to thalamic and brainstem nuclei and to spinal effectors. In this review, we address the functional role of the main PPTg outputs directed to the basal ganglia, thalamus, cerebellum and spinal cord. Together, the data that we discuss show that the PPTg may influence thalamocortical activity and spinal motoneuron excitability through its ascending and descending output fibers, respectively. Cerebellar nuclei may also relay signals from the PPTg to thalamic and brainstem nuclei. In addition to participating in motor functions, the PPTg participates in arousal, attention, action selection and reward mechanisms. Finally, we discuss the possibility that the PPTg may be involved in excitotoxic degeneration of the dopaminergic neurons of the substantia nigra through the glutamatergic monosynaptic input that it provides to these neurons.
This review argues neuronal mechanisms of postural control. Multi-sensory information such as somatosensory, visual, and vestibular sensation act on various areas of the brain so that adaptable postural control can be achieved. Automatic process of postural control, which is termed as postural reflexes including head–eye coordination accompanied by appropriate alignment of body segments, is mediated by the descending pathways from the brainstem. Cooperation of the vestibulospinal, reticulospinal, and tectospinal tracts contributes to this process. On the other hand, walking in unfamiliar circumstance requires cognitive process of postural control, which depends on knowledge of self-body, such as body schema, sense of postural verticality, and body motion in space. Such a bodily cognitive information is produced at the temporoparietal cortex. They are fundamental to sustention of vertical posture and construction of motor programs. The programs then run to execute anticipatory postural adjustment which is appropriate for achievement of goal-directed movements. The basal ganglia and cerebellum may affect both the automatic and cognitive processes of postural control through reciprocal connections with the brainstem and cerebral cortex, respectively. Consequently, impairments in cognitive function in addition to damages in the motor cortex, basal ganglia, and cerebellum may disturb appropriate postural control, resulting in falling.
Recently we incorporated an organotypic culture method in our basal ganglia research. In our preparation, we were successful in co-culturing more than two structures of interest1–3. This in vitro organotypic preparation combines the advantage of in vitro slice preparation for ease of intracellular sharp or patch recording under improved controlled experimental chemical environment with the in vivo preparation in which the structure of interest is not isolated from the source of their major afferents. In this report, we would like to present a triple culture preparation consisting of the tegmental pedunculopontine nucleus (PPN), the subthalamic nucleus (STN) and the substantia nigra (SN).
We examined the effects of a reversible blockade of pedunculopontine tegmental nucleus (PPN) in a monkey (macaca fuscata) which was trained to perform a lever-pull task with an arm. Neurons of PPN presented changes in their firing rate during arm movements. The changes were either an increase or a decrease. Muscimol (a γ-aminobutyric acid (GABA) agonist) was injected into PPN area to suppress its neuronal activity while the monkey was performing the task. The blockade of the activity of PPN neurons caused slowness of movements on the both sides of arms. The peak velocities of lever movement decreased and the reaction times increased. This work suggested that PPN had an excitatory effect to the initiation and the execution of voluntary movements of the limbs The monkey did not show any difficulty in performing the task and the success rates were kept high.
The inactivation of PPN might have resulted in a decreased activity of dopammergic neurons of the substantia nigra pars compacta. The possible pathophysiology of Parkinson’s disease regarding the decreased activity of the neurons of the PPN is discussed.
The present review was attempted to analyze the multiple channels of basal ganglia-thalamocortical connections, and the connections of their related nuclei. The prefrontal and motor areas consist of a number of modules, which seem to provide multiple subloops of the basal ganglia-thalamocortical connections in subhuman primates. There may be a gnat degree of convergence of the limbic, associative and motor loops at the level of the striatum, substantia nigra, pallidum, and the subthalamic nucleus as well as the pedunculopontine nucleus. Nigral dopaminergic neurons receive limbic input directly as well as indirectly through the striosomes in the striatum. Dopamine contributes to behavioral learning by signaling motivation and reinforcement. The pedunculopontine nucleus might be involved in behavioral state control, learning and reinforcement processes, locomotion and autonomic functions. Each subdivision of the motor areas receives a mixed and weighted transthalamic input from both the cerebellum and basal ganglia. In particular, based on the author's data, the hand/arm motor area and adjacent premotor area receive strong superficial basal ganglia-thalamocortical projections as well as the deep cerebello-thalamocortical projections. These areas, have very dense corticocotrical connections with other cortical areas, receive polymodal afferents from the parietal and temporal cortices, and integrated information, via multiple routes, from the prefrontal cortex. The author suggests that the ventrolateral part of the caudal medial pallidal segment (GPi) and the ventromedial part of the GPi are linked directly to these areas by ways of the oral part of ventral lateral nucleus (VLo) and the Ventral part of the parvicellular part of ventral anterior nucleus (VApc), respectively. These connections are thought to be involved in the acquisition and coordination of motor sequences.
Unlabelled:
The mesencephalic reticular formation (MRF) is formed by the pedunculopontine and cuneiform nuclei, two neuronal structures thought to be key elements in the supraspinal control of locomotion, muscle tone, waking, and REM sleep. The role of MRF has also been advocated in modulation of state of arousal leading to transition from wakefulness to sleep and it is further considered to be a main player in the pathophysiology of gait disorders seen in Parkinson's disease. However, the existence of a mesencephalic locomotor region and of an arousal center has not yet been demonstrated in primates. Here, we provide the first extensive electrophysiological mapping of the MRF using extracellular recordings at rest and during locomotion in a nonhuman primate (NHP) (Macaca fascicularis) model of bipedal locomotion. We found different neuronal populations that discharged according to a phasic or a tonic mode in response to locomotion, supporting the existence of a locomotor neuronal circuit within these MRF in behaving primates. Altogether, these data constitute the first electrophysiological characterization of a locomotor neuronal system present within the MRF in behaving NHPs under normal conditions, in accordance with several studies done in different experimental animal models.
Significance statement:
We provide the first extensive electrophysiological mapping of the two major components of the mesencephalic reticular formation (MRF), namely the pedunculopontine and cuneiform nuclei. We exploited a nonhuman primate (NHP) model of bipedal locomotion with extracellular recordings in behaving NHPs at rest and during locomotion. Different MRF neuronal groups were found to respond to locomotion, with phasic or tonic patterns of response. These data constitute the first electrophysiological evidences of a locomotor neuronal system within the MRF in behaving NHPs.
We present data from animal studies showing that the pedunculopontine tegmental nucleus-conserved through evolution, compartmentalized, and with a complex pattern of inputs and outputs-has functions that involve formation and updates of action-outcome associations, attention, and rapid decision making. This is in contrast to previous hypotheses about pedunculopontine function, which has served as a basis for clinical interest in the pedunculopontine in movement disorders. Current animal literature points to it being neither a specifically motor structure nor a master switch for sleep regulation. The pedunculopontine is connected to basal ganglia circuitry but also has primary sensory input across modalities and descending connections to pontomedullary, cerebellar, and spinal motor and autonomic control systems. Functional and anatomical studies in animals suggest strongly that, in addition to the pedunculopontine being an input and output station for the basal ganglia and key regulator of thalamic (and consequently cortical) activity, an additional major function is participation in the generation of actions on the basis of a first-pass analysis of incoming sensory data. Such a function-rapid decision making-has very high adaptive value for any vertebrate. We argue that in developing clinical strategies for treating basal ganglia disorders, it is necessary to take an account of the normal functions of the pedunculopontine. We believe that it is possible to use our hypothesis to explain why pedunculopontine deep brain stimulation used clinically has had variable outcomes in the treatment of parkinsonism motor symptoms and effects on cognitive processing. © 2016 International Parkinson and Movement Disorder Society.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder next to Alzheimer’s disease, affecting up to 1% of individuals aged 65–69 years and 3% of those over 80 years of age [1]. Among the cardinal features of parkinsonism (resting tremor, bradykinesia, rigidity, and postural instability), postural and gait disfunction leading to falls represents the largest single contributor to the number of emergency room visits and overall cost to the healthcare system relating to PD [2–4]. In addition, the fear of falling is associated with its recurrence, and frequently leads to a loss of independence and depression [5]. Postural and gait disfunction have proven particularly resistant to current dopamine and surgical therapies, which suggests a greater involvement of non-dopaminergic pathways and other brain loci distinct from the pallidal and subthalamic nuclei in their pathophysiology [6–14].
The lateral part of the mesopontine tegmentum contains functionally important structures involved in the control of posture and gait. Specifically, the mesencephalic locomotor region, which may consist of the cuneiform nucleus and pedunculopontine tegmental nucleus (PPN), occupies the interest with respect to the pathophysiology of posture-gait disorders. The purpose of this article is to review the mechanisms involved in the control of postural muscle tone and locomotion by the mesopontine tegmentum and the pontomedullary reticulospinal system. To make interpretation and discussion more robust, the above issue is considered largely based on our findings in the experiments using decerebrate cat preparations in addition to the results in animal experimentations and clinical investigations in other laboratories. Our investigations revealed the presence of functional topographical organizations with respect to the regulation of postural muscle tone and locomotion in both the mesopontine tegmentum and the pontomedullary reticulospinal system. These organizations were modified by neurotransmitter systems, particularly the cholinergic PPN projection to the pontine reticular formation. Because efferents from the forebrain structures as well as the cerebellum converge to the mesencephalic and pontomedullary reticular formation, changes in these organizations may be involved in the appropriate regulation of posture-gait synergy depending on the behavioral context. On the other hand, abnormal signals from the higher motor centers may produce dysfunction of the mesencephalic-reticulospinal system. Here we highlight the significance of elucidating the mechanisms of the mesencephalic-reticulospinal control of posture and locomotion so that thorough understanding of the pathophysiological mechanisms of posture-gait disorders can be made.
In Parkinson’s disease, there is evidence that dopaminergic (DA) neurons of the substantia nigra degenerate when they become electrically less active. Many non-DA structures including cholinergic neurons of the pedunculopontine nucleus (PPN) and orexinergic neurons of the hypothalamus, are also degenerating. Since these non-DA neurons are sources of excitatory inputs to the nigral DA neurons, their lesion in parkinsonian patients might play a key role in the progression of DA neuronal death. The first goal of this study was to evaluate the effect of a cholinergic PPN lesion on the survival of nigral DA neurons in healthy and parkinsonian rats and macaques. We found that 1) a PPN cholinergic lesion induced neuronal atrophy and death; 2) a DA lesion alone resulted in a loss of PPN cholinergic neurons; and 3) adding a PPN cholinergic lesion to a DA lesion in rats when the process of DA degeneration was in progress exacerbated neuronal losses in both systems. Last, the rate of DA degeneration was highly correlated to the level of cholinergic loss. Our results highlight the key role of the PPN in the physiopathology of Parkinson’s disease and clearly demonstrate strong reciprocal interactions with nigral DA neurons. The second aim of our study was to focus on the hypothalamic orexinergic system using a morphological approach in macaques. We show that orexinergic fibers are in position to modulate DA neurons activity. However, a relatively selective DA lesion in macaques was not sufficient to induce death of the orexinergic neurons. These data suggest that the loss of orexinergic neurons observed in parkinsonian patients likely results from non-DA lesions.
Artículo de revisión RESUMEN El núcleo pedúnculo pontino (NPP) es una estructura heterogénea desde el punto de vista celular, bioquímico y funcional. Forma parte de la región mesencefálica motora de forma extensa y se ha demostrado a través de estudios en modelos animales su participación en iniciación y mantenimiento del movimiento. Desde el punto de vista terapéutico se ha encontrado que los modelos animales de enfermedad de Parkinson presentan mejoría con activación y empeoran con la inhibición de este núcleo. Recién se ha realizado las primeras intervenciones quirúrgicas sobre esta estructura en seres humanos, con ellas se ha obtenido información acerca del registro eléctrico y respuesta de los pacientes con la estimulación eléctrica. Para la localización de esta estructura se cuenta en la actualidad con apoyo de estudios de imagen, atlas de estereotaxia, uso de microrregistro transoperatorio, estimulación y valoración del paciente durante dicho procedimiento. Todos estos recursos de localización deben ser utilizarse de manera conjunta para mejorar resultados y disminuir al mínimo los riesgos de este procedimiento. Ahora, la intervención sobre este núcleo se vislumbra como una posibilidad de tratamiento de síntomas axiales resistentes a L-dopa en pacientes con enfermedad de Parkinson y otros síndromes parkinsónicos. Palabras clave: enfermedad de Parkinson, estereotáxia, núcleo pedúnculo pontino, estimulación cerebral profunda. Stereotaxic method to locate the pedunculo pontine nucleus (PPN) in human beings ABSTRACT From the cellular, biochemical and functional point of view the pedunculopontine nucleus (PPN) is an heterogeneous structure. The PPN is part of the «mesencephalic motor region» and it has been found to participate in initiating and maintaining movement. From the therapeutic point of view, it has been found that Parkinson's disease animal models show improvement with activation and worsening with inhibition of this nucleus. Only recently have some neurosurgical teams made the first surgical interventions on this structure on human beings, and with these interventions gathered information about registering and clinical response to electrical stimulation during surgery. To locate this structure, we have the aid of images, stereotaxic atlases, micro registering and clinical response of the patient during surgery. All of these tools must be used in a cooperative fashion in order to obtain the best clinical response and to reduce risk as much as possible. Today, the intervention on this nucleus is seen as a possible therapy for L-Dopa resistant axial symptoms in patients with Parkinson's disease and other parkinsonic syndromes.
El núcleo pedúnculopóntico (NPP) se encuentra localizado en el tegmento pontomesencefálico en su región dorsolateral. Este núcleo es un complejo de neuronas colinérgicas y nocolinérgicas que por su situación anatómica y sus numerosas conexiones con estructuras como los ganglios de la base, juega un papel importante en la producción y la modulación del movimiento, aspecto que lo involucra en la fisiopatología de la Enfermedad de Parkinson. Estudios post-mortem en pacientes que padecieron enfermedad de Parkinson, mostraron una significativa degeneración del NPP. También se han explicado las manifestaciones clínicas de la Enfermedad del Parkinson, desde la disfunción del núcleo, y se ha propuesto la estimulación cerebral profunda del mismo como parte de la terapia de la Enfermedad de Parkinson. Este artículo de revisión, pretende explorar el papel fisiopatológico y funcional del NPP.
Fixational saccades are small, involuntary eye movements that occur during attempted visual fixation. Recent studies suggested that several cognitive processes affect the occurrence probability of fixational saccades. Thus, there might be an interaction between fixational saccade-related motor signals and cognitive signals. The pedunculopontine tegmental nucleus (PPTN) in the brainstem has anatomical connections with numerous saccade-related and limbic areas. Previously, we reported that a group of PPTN neurons showed transient phasic bursts or a pause in activity during large visually guided and spontaneous saccades, and also showed sustained tonic changes in activity with task context. We hypothesised that single PPTN neurons would relay both fixational saccade-related and task context-related signals, and might function as an interface between the motor and limbic systems. We recorded the activity of PPTN neurons in behaving monkeys during a reward-biased task, and analysed neuronal activity for small fixational saccades during visual fixation, and compared it with the activity for large visually guided targeting saccades and large spontaneous saccades during intertrial intervals. A population of PPTN neurons exhibited a fixational saccade-related phasic increase in activity, and the majority of them also showed activity modulation with large targeting saccades. In addition, a group of these neurons showed a task-related tonic increase in activity during the fixation period, and half of them relayed the saccade signal only when the neuron exhibited higher tonic activity during the task execution period. Thus, fixational saccade-related signals of PPTN neurons overlap with tonic task-related signals, and might contribute to the cognitive modulation of fixational saccades.
I review substrates for the execution of normal gait and to understand pathophysiological mechanisms of gait failure in basal ganglia dysfunctions. In Parkinson's disease, volitional and emotional expressions of movement processes are seriously affected in addition to the disturbance of automatic movement processes, such as adjustment of postural muscle tone and rhythmic limb movements during walking. These patients also suffer from muscle tone rigidity and postural instability, which may also cause reduced walking capabilities. Clinical and neurophysiological studies have suggested the importance of basal ganglia connections with the cerebral cortex and limbic system in the expression of volitional and emotional behaviors, respectively. On the other hand, basal ganglia output toward the brainstem (basal ganglia-brainstem system) can be critically involved in the integrative control of muscle tone and locomotion. Recently emphasis has been placed on the importance of body schema, or internal postural model, which is generated at parietal cortex by integrating multimodal (proprioceptive, visual, auditory and vestibular) sensory inputs. The body schema can be required for motor programming of intentionally controlled precise movements and anticipatory postural control. Based on these considerations I provide a hypothetical model for understanding the role of the basal ganglia in the control of volitional and automatic aspects of movements and pathophysiological mechanisms of basal ganglia motor disorders.
Previous investigations in various motor and sensory cortical areas have shown that fast oscillations (20-80 Hz) of focal electroencephalogram and multiunit activities occur spontaneously during increased alertness or are dependent upon optimal sensory stimuli. We now report the presence of 20- to 40-Hz rhythmic activities in intracellularly recorded thalamocortical cells of the cat. In some neurons, subthreshold oscillations were triggered by depolarizing pulses and eventually gave rise to action potentials. In other neurons, the oscillations consisted of fast prepotentials, occasionally generating full spikes that arose from the resting or even from hyperpolarized membrane potential levels, and leading to trains of spikes at more depolarized levels. The rhythmic nature of these fast prepotentials was confirmed by means of an autocorrelation study, which demonstrated clear peaks at 25-ms intervals (40 Hz). In view of the recent evidence that mesopontine cholinergic nuclei trigger and maintain activation processes in thalamocortical systems, we tested the possibility that stimulation of these brainstem nuclei potentiates the 40-Hz waves on the background of the cortical electroencephalogram. This was indeed the case. The potentiation outlasted the stimulation by 10-20 s. The brainstem-induced facilitation of cortical 40-Hz oscillations was blocked by scopolamine, a muscarinic antagonist. That this facilitation was transmitted by brainstem-thalamic cholinergic projections was confirmed by persistence of the phenomenon after large excitotoxic lesions of the nucleus basalis of Meynert.
The only mesopontine neurons previously described as involved in the transfer of ponto-geniculo-occipital (PGO) waves from the brain stem to the thalamus were termed PGO-on bursting cells. We have studied, in chronically implanted cats, neuronal activities in brain-stem peribrachial (PB) and laterodorsal tegmental (LDT) cholinergic nuclei in relation to PGO waves recorded from the lateral geniculate (LG) thalamic nucleus during rapid-eye-movement (REM) sleep. We constructed peri-PGO histograms of PB/LDT cells' discharges and analyzed the interspike interval distribution during the period of increased neuronal activity related to PGO waves. Six categories of PGO-related PB/LDT neurons with identified thalamic projections were found: 4 classes of PGO-on cells: PGO-off but REM-on cells: and post-PGO cells. The physiological characteristics of a given cell class were stable even during prolonged recordings. One of these cell classes (1) represents the previously described PGO-on bursting neurons, while the other five (2-6) are newly discovered neuronal types. (1) Some neurons (16% of PGO-related cells) discharged stereotyped low-frequency (120-180 Hz) spike bursts preceding the negative peak of the LG-PGO waves by 20-40 msec. These neurons had low firing rates (0.5-3.5 Hz) during all states. (2) A distinct cell class (22% of PGO-related neurons) fired high-frequency spike bursts (greater than 500 Hz) about 20-40 msec prior to the thalamic PGO wave. These bursts were preceded by a period (150-200 msec) of discharge acceleration on a background of tonically increased activity during REM sleep. (3) PGO-on tonic neurons (20% of PGO-related neurons) discharged trains of repetitive single spikes preceding the thalamic PGO waves by 100-150 msec, but never fired high-frequency spike bursts. (4) Other PGO-on neurons (10% of PGO-related neurons) discharged single spikes preceding thalamic PGO waves by 15-30 msec. On the basis of parallel intracellular recordings in acutely prepared, reserpine-treated animals, we concluded that the PGO-on single spikes arise from conventional excitatory postsynaptic potentials and do not reflect tiny postinhibitory rebounds. (5) A peculiar cellular class, termed PGO-off elements (8% of PGO-related neurons), consisted of neurons with tonic, high discharge rates (greater than 30 Hz) during REM sleep. These neurons stopped firing 100-200 msec before and during the thalamic PGO waves. (6) Finally, other neurons discharged spike bursts or tonic spike trains 100-300 msec after the initially negative peak of the thalamic PGO field potential (post-PGO elements, 23% of PGO-related neurons).(ABSTRACT TRUNCATED AT 400 WORDS)
This study was performed to examine the hypothesis that thalamic-projecting neurons of mesopontine cholinergic nuclei display activity patterns that are compatible with their role in inducing and maintaining activation processes in thalamocortical systems during the states of waking (W) and rapid-eye-movement (REM) sleep associated with desynchronization of the electroencephalogram (EEG). A sample of 780 neurons located in the peribrachial (PB) area of the pedunculopontine tegmental nucleus and in the laterodorsal tegmental (LDT) nucleus were recorded extracellularly in unanesthetized, chronically implanted cats. Of those neurons, 82 were antidromically invaded from medial, intralaminar, and lateral thalamic nuclei: 570 were orthodromically driven at short latencies from various thalamic sites: and 45 of the latter elements are also part of the 82 cell group, as they were activated both antidromically and synaptically from the thalamus. There were no statistically significant differences between firing rates in the PB and LDT neuronal samples. Rate analyses in 2 distinct groups of PB/LDT neurons, with fast (greater than 10 Hz) and slow (less than 2 Hz) discharge rates in W, indicated that (1) the fast-discharging cell group had higher firing rates in W and REM sleep compared to EEG-synchronized sleep (S), the differences between all states being significant (p less than 0.0005); (2) the slow-discharging cell group increased firing rates from W to S and further to REM sleep, with significant difference between W and S (p less than 0.01), as well as between W or S and REM sleep (p less than 0.0005). Interspike interval histograms of PB and LDT neurons showed that 75% of them have tonic firing patterns, with virtually no high-frequency spike bursts in any state of the wake-sleep cycle. We found 22 PB cells that discharged rhythmic spike trains with recurring periods of 0.8-1 sec. Autocorrelograms revealed that this oscillatory behavior disappeared when their firing rate increased during REM sleep. Dynamic analyses of sequential firing rates throughout the waking-sleep cycle showed that none of the full-blown states of vigilance is associated with a uniform level of spontaneous firing rate. Signs of decreased discharge frequencies of mesopontine neurons appeared toward the end of quiet W, preceding by about 10-20 sec the most precocious signs of EEG synchronization heralding the sleep onset. During transition from S to W, rates of spontaneous discharges increased 20 sec before the onset of EEG desynchronization.(ABSTRACT TRUNCATED AT 400 WORDS)
The distribution of acetylcholine neurons in the brainstem of the cat was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared to that of catecholamine neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. The largest group of ChAT-positive (+) neurons was located in the lateral pontomesencephalic tegmentum within the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus rostrally and within the parabrachial nuclei and locus coeruleus nucleus more caudally. TH+ neurons were found to be coextensive and intermingled with ChAT+ neurons in the dorsolateral pontomesencephalic tegmentum, where the number of ChAT+ cells (approximately 18,500) exceeded that of the TH+ cells (approximately 12,000). In the caudal pons, scattered ChAT+ neurons were situated in the ventrolateral tegmentum together with TH+ neurons. In the medulla, numerous ChAT+ cells were located in the lateral tegmental field, where they extended in a radial column from the dorsal motor nucleus of the vagus to the ventrolateral tegmentum around the facial and ambiguus nuclei, occupying the position of preganglionic parasympathetic neurons of the 7th, 9th, and 10th cranial nerves. TH+ cells were also present in this field. Neurons within the general visceral, special visceral, and somatic motor cranial nerve nuclei were all immunoreactive to ChAT. Scattered ChAT+ neurons were also present within the medullary gigantocellular and magnocellular tegmental fields together with a small number of TH+ neurons. Other groups of ChAT+ cells were identified within the periolivary nuclei, parabigeminal nucleus, prepositus hypoglossi nucleus, and the medial and inferior vestibular nuclei. Acetylcholine neurons thus constitute a heterogeneous population of cells in the brainstem, which in addition to including the somatic and visceral efferent systems, comprises many other discrete systems and represents an important component of the brainstem reticular formation. The proximity to and interdigitation with catecholamine neurons within these systems may be of important functional significance.
This article reviews the electroresponsive properties of single neurons in the mammalian central nervous system (CNS). In
some of these cells the ionic conductances responsible for their excitability also endow them with autorhythmic electrical
oscillatory properties. Chemical or electrical synaptic contacts between these neurons often result in network oscillations.
In such networks, autorhythmic neurons may act as true oscillators (as pacemakers) or as resonators (responding preferentially
to certain firing frequencies). Oscillations and resonance in the CNS are proposed to have diverse functional roles, such
as (i) determining global functional states (for example, sleep-wakefulness or attention), (ii) timing in motor coordination,
and (iii) specifying connectivity during development. Also, oscillation, especially in the thalamo-cortical circuits, may
be related to certain neurological and psychiatric disorders. This review proposes that the autorhythmic electrical properties
of central neurons and their connectivity form the basis for an intrinsic functional coordinate system that provides internal
context to sensory input.
The efferent connections of the paramedian pontine reticular formation have been studied in the cat in autoradiographic experiments designed to analyze direct and indirect preoculomotor pathways. Injections of tritium-labelled amino acids were placed (1) near the border between the oral and caudal subdivisions of the nucleus pontis centralis, (2) in more rostral and dorsal parts of the pontine tegmentum, (3) at the pontomesencephalic border, and (4) at the pontomedullary border.
Tegmental injections of the first group were unique in labelling a direct ipsilateral pathway to the abducens nucleus and nucleus prepositus hypoglossi. More rostral injections failed to produce discrete labelling of the nuclei of the extraocular muscles but labelled nearby tegmentum and central gray substance. Caudal deposits, involving the pontomedullary reticular formation at its junction with the abducens, perihypoglossal and vestibular nuclei, labelled a decussating fiber system reaching the contralateral abducens nucleus, nucleus prepositus hypoglossi and parts of the vestibular complex. In a single additional case, an injection placed in the oculomotor complex produced heavy labelling of the abducens nuclei.
All tegmental injections labelled discrete reticulo-reticular and other variably complex longitudinal pathways. Most injections of (a) the pontomedullary and (b) the pontomesencephalic zones elicited labelling of the pretectum including the nucleus of the optic tract. An incidental finding in the latter group was dense labelling of the pars compacta of the substantia nigra, subthalamic nucleus, and (1 case) entopeduncular nucleus; in one case of each of these groups, labelled fibers were traced to the external pallidum.
These observations suggest that, with respect to its efferent oculomotor affiliations, the paramedian pontine tegmentum may be divided into compartments whose supranuclear connections are distinct but for the most part heavily weighted toward influencing the abducens nucleus and periabducens region. Considered within the framework of behavioral and physiological studies of the so-called pontine gaze center, and studies of pontine afferents, the findings are interpreted as suggesting a functional differentiation of these tegmental zones with respect to their influence on eye-head coordination.
Intrapontine microinjections of serotonin in acutely decerebrated cats resulted in the bilateral augmentation of the postural muscle tone of the hindlimbs. Optimal injection sites were located in the dorsomedial part of the rostral pontine reticular formation corresponding to the nucleus reticularis ponds oralis (NRPo). In this study, attempts were made to elucidate the cellular basis for the serotoninergically induced augmentation of postural muscle tone by recording the electromyographic (EMG) activity of hindlimb extensor muscles, the monosynaptic reflex responses evoked by electrical stimulation of group Ia muscle afferent fibres and the membrane potentials of hindlimb alpha-motoneurons (MNs). Serotonin injections resulted not only in the augmentation of the EMG activity of gastrocnemius soleus muscles, but also in the restoration of EMG suppression, which was induced by previous injection of carbachol into the NRPo. Extensor and flexor monosynaptic reflex responses were facilitated by serotonin injections into the NRPo. Such reflex facilitation was not induced by serotonin injections into the mesencephalic or the medullary reticular formation. Intrapontine serotonin injections resulted in membrane depolarization of extensor and flexor MNs with decreases in input resistance and rheobase. Spontaneous depolarizing synaptic potentials (EPSPs) increased in both frequency and amplitude. Peak voltage of Ia monosynaptic EPSPs also increased. Serotonin injections which followed carbachol injections resulted in membrane depolarization of MNs along with an increase in the frequency of spontaneous EPSPs and a decrease in carbachol-induced inhibitory postsynaptic potentials. Following pontine carbachol injections, antidromic and orthodromic responses in MNs were suppressed. Discharges of MNs evoked by intracellular current injections were also suppressed, but were restored following serotonin injections. These results indicate that postsynaptic excitation, presynaptic facilitation and disinhibition (withdrawal of postsynaptic inhibition) simultaneously act on the hindlimb MNs during serotonin-induced postural augmentation and restoration.
The efferent connections of the brain stem nucleus tegmenti pedunculopontinus were studied in the rat using the techniques of anterograde and retrograde transport of the enzyme horseradish peroxidase, laying particular emphasis on that part of pedunculopontinus which receives direct descending projections from the basal ganglia and related nuclei. In a preliminary series of experiments horseradish peroxidase was injected into either the entopeduncular nucleus or the subthalamic nucleus and, following anterograde transport of enzyme, terminal labelling was identified in nucleus tegmenti pedunculopontinus, surrounding the brachium conjunctivum in the caudal mesencephalon.
A newly discovered class of neurons, ponto-geniculo-occipital (PGO) burst neurons, has PGO wave relationships of phase-leading, stereotyped discharge bursts, and the highest reported discharge specificity and coherence; these neurons thus fulfill correlative criteria for output generator neurons for PGO waves. The PGO burst neurons are recorded in a discrete dorsal brainstem area in apposition to the brachium conjunctivum.
Muscular atonia and cortical desynchronization, two signs of desynchronized sleep, can be enhanced or suppressed by direct injection of carbachol into the pontine brain stem of cats. The positive effects are graded, being maximal in the gigantocellular tegmental field and less marked in adjacent nuclei. These positive effects are dose-dependent. Suppressive effects of carbachol are maximal in the region of the locus coeruleus and are dose-dependent but do not exceed those of the vehicle alone. The results support the hypothesis that cholinergic mechanisms of the pontine tegmentum are involved in desynchronized sleep generation.
Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH-diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents.
Choline acetyltransferase immunhistochemistry was employed at light and electron microscopic levels in order to determine the distribution of cholinergic neurons in two subdivisions of the rat pedunculopontine tegmental nucleus that were previously defined on cytoarchitectonic grounds, and to compare the synaptic inputs to cholinergic and non-cholinergic somata in the subnucleus dissipatus, which receives major input from the substantia nigra. Large cholinergic neurons were found in both the pars compacta and the pars dissipata of the pedunculopontine nucleus. However, they were intermingled with non-cholinergic neurons and did not respect the cytoarchitectural boundaries of the nucleus. Ultrastructural study showed that all cholinergic neurons in the subnucleus dissipatus exhibited similar features. The majority had large somata (largest diameter ⩾20 μm) containing abundant cytoplasmic organelles and nuclei displaying a few shallow invaginations. Synaptic terminals on the cholinergic cell bodies were scarce and unlabeled boutons containing spherical synaptic vesicles and establishing asymmetric synaptic junctions were the dominant type. In contrast, the non-cholinergic neurons presented prominent differences in the size of their somata as well as in the distribution of axosomatic synapses. Two almost equally represented classes of non-cholinergic neurons which are referred to as large (largest diameter ⩾20 μm) and small (largest diameter
In order to determine whether the cholinergic fibres that innervate the substantia nigra make synaptic contact with dopaminergic neurons of the substantia nigra pars compacta, a double immunocytochemical study was carried out in the rat and ferret. Sections of perfusion-fixed mesencephalon were incubated first to reveal choline acetyltransferase immunoreactivity to label the cholinergic terminals and then tyrosine hydroxylase immunoreactivity to label the dopaminergic neurons. Each antigen was localized using peroxidase reactions but with different chromogens.
The termination of the substantia nigra pars reticulata efferents in the nucleus tegmenti pedunculopontinus was studied in the rat by using the anterograde tracer Phaseolusvulgaris ‐leucoagglutinin (PHA‐L). Both large and small injections of PHA‐L in various portions of the substantia nigra pars reticulata labeled varicose fibers in the ipsilateral and contralateral nucleus tegmenti pedunculopontinus, subnucleus dissipatus as well as in the ipsilateral nucleus tegmenti pedunculopontinus, subnucleus compactus. However, the bulk of the nigral fibers appeared to terminate in the medial two‐thirds of the ipsilateral subnucleus dissipatus of the pedunculopontine nucleus and exhibited a discrete dorsoventral topographical pattern. The terminal plexus displayed patches of uneven density, which was partly due to the numerous fiber bundles passing through the pedunculopontine nucleus, but also to an obvious preference of nigral fibers for some cells. Electron microscopic examination confirmed that nearly all of the varicosities observed in the light microscope contained synaptic vesicles and represented either terminal boutons or boutons en passant. The labeled boutons were elongated (average length: 1.5 μm) and consistently contained a prominent group of mitochondria.
The results suggest that the nigral input to the nucleus tegmenti pedunculopontinus may be directed toward specific subpopulation(s) of pedunculopontine neurons and may influence not only cells in the subnucleus dissipatus, but also in the subnucleus compactus.
Responses of 43 pedunculopontine area (PPN area) neurons to electrical stimulation of the substantia nigra (SN) were studied in anesthetized rats. An intracellular recording technique was used to demonstrate that SN stimulation evoked hyperpolarizing potentials, which were identified by intracellular injections as inhibitory postsynaptic potentials (IPSPs). These IPSPs were often followed by a rebound depolarization that originates several spike potentials. These IPSPs were characterized as monosynaptic, with latencies varying from 1.0 to 8.5 ms. Similar results were observed in some animals with chronic unilateral coronal lesion just rostral to subthalamic nucleus (STH), which severed the rostral afferents. PPN are neurons were also antidromically activated by SN stimulation. Two PPN area projection neurons were clearly identified. Mean latency of one group was 0.71 ms; mean latency of the second group was 5.16 ms. The morphological analysis of a neuron inhibited by SN stimulation and labeled with horseradish peroxidase (HRP) demonstrated that the soma was fusiform in shape, with the axon originating in the soma and collaterals and a large dendritic field extending in the ventrodorsalis direction. The results indicate that the PPN area is reciprocally connected with the SN, which elicits an inhibitory effect on PPN area neurons.
For the last decade the functional organization pf cholinergic neurons has dominated studies of the basal forebrain. Cholinergic neurons in the brain, exclusive of motor neurons and interneurons, are found in two spatially separate groups (Armstrong et al., 1983, Mesulam et al., 1984). The rostral group, located in the basal forebrain, has received substantial attention because of its corticopedal projections (Mesulam et al., 1984) and its’ degeneration in Alzheimer’s disease (Coyle et al., 1983). The caudal group is found in the laterodorsal tegmental nucleus (LDT) and pedunculopontine nucleus (PPT) within the pontine tegmentum (Vincent et al., 1983; Mesulam et al., 1984; Satoh and Fibiger, 1986), and is the source of cholinergic innervation to the basal forebrain, thalamus and brainstem (Sofroniew et al., 1985; Hallenger et al., 1987; Maley et al., 1988; Rye et al., 1988; Jones, 1990).
Carbachol, a long-acting cholinergic agonist, was microinjected (4 micrograms/250 nl per 90 s) into 90 sites within the anterodorsal pontine tegmentum of four cats and the time to onset and percentage of time spent in a desynchronized sleep-like state during 40 min postinjection were calculated. Compared with more posteroventral pontine sites, the shorter latencies and higher percentages observed confirmed earlier predictions of a sensitive cholinoceptive zone in the anterodorsal pons. In 27 trials a desynchronized sleep-like state was observed within 5 min; in 31 trials the latency was 5-10 min and in the remaining 32 trials, greater than 10 min. Plotting the desynchronized sleep-like state latency and the desynchronized sleep-like state percentage as a function of the three-dimensional coordinates revealed that injection sites with short latency (less than 5 min) and high percentage (greater than 80%) were concentrated between the coordinates of P 1.0 to 3.5 and V -3.5 to -5.5, at the lateral coordinate L 2.0. On the frontal plane, the short desynchronized sleep-like state latency and high desynchronized sleep-like state percentage sites begin in the pontine tegmental region just lateral to the ventral tegmental nucleus and extend 3 mm ventrocaudally. A regression plot of the data in sagittal plane 2.0 revealed a short latency axis, around which the short latency sites cluster, running in a slightly dorsoventral direction from about P 1.0 to V -4.0 to P 4.0 to V -5.5. This observation suggests that the sensitive zone might approximate a cylinder in shape, a hypothesis supported by the correlation of longer latencies and lower percentages at increasing radial distance from the axis. The non-linear relationship between cholinergic potency and distance from the short latency axis suggests that the desynchronized sleep-like state latency is a function of two factors; a variable diffusion-based delay of carbachol to distant neuronal populations involved in the desynchronized sleep-like state production, and a fixed recruitment-based delay following activation of neurons in the sensitive zone. Interpretation of these findings in light of earlier studies involving microstimulation of the pontine tegmentum argue in favor of a distributed network of discrete neuronal populations as the source of desynchronized sleep generation.
Microinjections of the cholinergic agonist carbachol into a caudal part of the pontine reticular formation of the rat induce a rapid eye movement sleep-like state. This carbachol-sensitive region of the pontine reticular formation is innervated by cholinergic neurons in the pedunculopontine and laterodorsol tegmental nuclei. The same population of cholinergic neurons also project heavily to the thalamus, where there is good evidence that acetylcholine facilitates sensory transmission and blocks rhythmic thalamocortical activity. The present study was undertaken to examine the degree to which single cholinergic neurons in the mesopontine tegmentum project to both the carbachol-sensitive region of the pontine reticular formation and the thalamus, by combining double fluorescent retrograde tracing and immunofluorescence with a monoclonal antibody to choline acetyltransferase in the rat. The results indicated that a subpopulation (5-21% ipsilaterally) of cholinergic neurons in the mesopontine tegmentum projects to both the thalamus and the carbachol-sensitive site of the pontine reticular formation, and these neurons represented the majority (45-88%) of cholinergic neurons projecting to the pontine reticular formation site. The percentage of cholinergic neurons with dual projections was higher in the pedunculopontine tegmental nucleus (6-27%) than in the laterodorsal tegmental nucleus (4-11%). In addition, mixed with cholinergic neurons in the mesopontine tegmentum, there was a small population of dually projecting neurons that did not appear to be cholinergic. Mesopontine cholinergic neurons with dual projections may simultaneously modulate neuronal activity in the pontine reticular formation and the thalamus, and thereby have the potential of concurrently regulating different aspects of rapid eye movement sleep.
Membrane properties and postsynaptic responses to stimulation of the substantia nigra reticulata (SNr) of the neurons in rat pedunculopontine nucleus (PPN) were studied in an in vitro parasagittal slice preparation using intracellular recording techniques. Based on electrical membrane properties, PPN neurons were classified into 3 types (types I, II and II). The unique feature of the type I neuron was the low threshold calcium spike while the type II neuron had various inward and outward rectifications. The type III neuron showed no such features as those observed in type I or II neurons. Some recorded neurons were intracellularly labeled with biocytin to study their morphology, and their transmitter phenotype was investigated by immunocytochemistry for choline acetyltransferase (ChAT). The type I and III neurons were found to be non-cholinergic, but 50% of the labeled type II neurons were immunopositive for ChAT. Morphological features of type II neurons were also different from type I or III neurons. The soma of the type II neuron was almost always more than twice as large as that of type I and III neurons. Inhibitory postsynaptic potentials (IPSPs) were induced in all 3 types of PPN neurons following stimulation of SNr. SNr-induced IPSPs were usually followed by a slow depolarizing potential from which rebound spikes were triggered. These rebound excitations were found only in type I and II neurons. These data indicate that heterogeneous groups of neurons exist in the PPN in terms of morphology, transmitter phenotypes and electrical membrane properties.
Studies of the pedunculopontine (PPT) and laterodorsal tegmental (LDT) nuclei in the mesopontine tegmentum have emphasized the organization and projections of the cholinergic neurons. We report here that exhibiting glutamate immunoreactivity are present in both the LDT and PPT. These glutamatergic neurons are interspersed among the cholinergic neurons within both nuclei with no apparent segregation. These data raise the possibility that excitatory amino acids contribute to the functions of the LDT and PPT.
A map of cholinergic cells of the human brainstem identified by immunohistochemistry of choline acetyltransferase (ChAT) is presented, along with a map of acetylcholinesterase (AChE)-containing cells and fibers. ChAT-positive structures belong to 4 brainstem systems: the cranial motor nuclei; the parabrachial complex; the reticular system; and the vestibular system. All motor nuclei of the cranial nerves, as well as the nucleus supraspinalis, are ChAT-positive. The positively staining structures of the parabrachial system include the nucleus tegmentali pedunculopontinus, and the nuclei parabrachialis medialis and lateralis. Nuclei of the reticular system containing some ChAT-positive cells include the nucleus reticularis pontis oralis and caudalis, the nucleus reticularis tegmenti pontis, the nucleus reticularis gigantocellularis, the nucleus reticularis lateralis and the formatio reticularis centralis (medulla). Structures of the vestibular and auditory systems which contain some ChAT-positive cells include the nucleus vestibularis lateralis, and the nuclei olivaris superioris medialis and lateralis. All ChAT-positive structures stain strongly for AChE. AChE-positive, ChAT-negative structures were noted in several sensory systems. The substantia nigra, locus coeruleus and raphe nuclei, known to contain non-cholinergic cells, also stain positively. The significance of the AChE-positive, ChAT-negative staining in most structures remains to be determined. A knowledge of the cholinergic systems of human brain may be important to an understanding of the pathology of a number of diseases.
Atriopeptin, the atrial natriuretic peptide, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte balance. Immunohistochemical studies have shown that large, multipolar atriopeptin-like immunoreactive (APir) neurons are present in areas of the midbrain corresponding to the large neurons of the pedunculopontine tegmental (PPT) and lateral dorsal tegmental (TLD) nuclei, all of which can be stained immunohistochemically for choline acetyltransferase-like immunoreactivity (ChATir). A subpopulation of these cholinergic PPT and TLD neurons are also known to contain substance P-like immunoreactivity (SPir). Using an immunofluorescent technique that allows simultaneous localization of two antigens, we have studied the relationship between APir, SPir and ChATir in the pontine tegmentum of the rat. We have found that the large multipolar APir neurons of the pontine tegmentum are identical to the ChATir neurons of the PT and TLD nuclei and a subpopulation of the APir neurons in PPT and TLD neurons are also SPir.
A substantial population of cells from the nucleus tegmenti pedunculopontinus was demonstrated to have descending projections to the spinal cord using fluorescent retrograde axonal tracers. Double-labeling studies showed that separate perikarya in this region have descending versus ascending projections. The distributions of the cell bodies with ascending or descending projections were spatially distinct, but partially overlapping. Some ascending, but not the descending, projections were cholinergic.
Previous studies have suggested that the pedunculopontine tegmental nucleus (PPTn) is reciprocally connected with extrapyramidal motor system nuclei (EPMS) whereas other studies have implicated the PPTn in behavioral state control phenomena such as sleep-wakefulness cycles. Many of these studies define the nonprimate PPTn as an area of mesopontine tegmentum which is labeled from injections of anterograde tracers into the basal ganglia. Recently, we have defined the rat PPTn as a large-celled, cholinergic nucleus. The rat PPTn is cytologically distinct from a group of smaller, noncholinergic neurons that are medially adjacent to the PPTn. This noncholinergic group is further distinguished from the PPTn by its afferent input from the globus pallidus, entopeduncular nucleus, and substantia nigra. We refer to the latter area as the midbrain extrapyramidal area (MEA).
Using combined choline acetyltransferase immunohistochemistry of the PPTn and WGA-HRP retrograde tracing from the EPMS, we investigated the efferent connections of the MEA and PPTn to the EPMS in the rat. The noncholinergic MEA, rather than the PPTn, is the major source of tegmental innervation to the globus pallidus, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra, and motor cortex. In contrast, the cholinergic PPTn is the major source of tegmental innervation to the ventrolateral thalamic nucleus. This finding is in contradistinction to thalamic projections from the surrounding reticular formation, which are identified only after WGA-HRP injections into “nonspecific” thalamic nuclei. This body of evidence suggests that the noncholinergic MEA represents an additional component of the EPMS and may correspond to the “mesencephalic locomotor region.” The cholinergic PPTn may play a role in more global thalamic functions such as the “reticular activating system” rather than a primary role in motor function.
Ascending projections from the pedunculopontine tegmental nucleus (PPT) and the surrounding mesopontine tegmentum to the forebrain in the rat are here examined by using both retrograde and anterograde tracing techniques combined with choline acetyltransferase (ChAT) immunohistochemistry. The anterogradely transported lectin Phaseolus vulgaris-leukoagglutinin (PHA-L) was iontophoretically injected into the PPT in 12 rats. Anterogradely labelled fibers and varicosities were observed in the thalamic nuclei, confirming the findings of our previous retrograde studies (Hallanger et al: J. Comp. Neurol. 262:105–124, ′87). In addition, PHA-L-labelled fibers and varicosities suggestive of terminal fields were observed in the anterior, tuberal, and posterior lateral hypothalamic regions, the ventral pallidum in the region of the nucleus basalis of Meynert, the dorsal and intermediate lateral septal nuclei, and in the central and medial nuclei of the amygdala.
The distribution and collaterlization of ascending and descending projections from neurons in the nucleus tegmenti pedunculopontinus (PPN) were studied in the rat by using retrograde transport of HRP, HRP/WGA, and fluorescent dyes. The PPN and its two subdivisions, the subnucleus compactus (PPNc) and subnucleus dissipatus (PPNd), were delineated on sagittal Nisslstained sections by using cytoarchitectural features as guidelines.
Large bilateral pressure injections of HRP and/or fluorescent dyes into the cervical cord retrogradely labeled moderate numbers of fusiform and polygonal PPN cells which ranged in size between 65 and 390 μm2. The labeled cells were scattered throughout the PPNd and were somewhat more numerous in the medial half of the subnucleus. The PPNc contained only occasional labeled cells in its ventralmost portion.
Following single unilateral HRP/WGA injections in the striatum, globus pallidus, entopeduncular nucleus, subthalamus, or the substantia nigra, the distribution of the labeled cells was similar to that of the spinal cord-projecting PPN neurons. Multiple HRP injections were then made bilaterally in the substantia nigra and the entopeduncular nucleus and/or subthalamus in order to label the entire population of PPN neurons projecting to the basal ganglia. In this case, not only the PPNd but also the PPNc contained a substantial number of retrogradely labeled cells. The rostrally projecting PPN cells outnumbered 5.4 times those projecting to the spinal cord, and their somata were somewhat larger, ranging between 114 and 472 μm2. While both fusiform and polygonal shapes were encountered, the polygonal cell somata were more numerous.
In the double-labeling experiments, Granular Blue and Diamidino Yellow Dihydrochloride were injected into the cervical cord and the entopeduncular nucleus or subthalamus. In general, these experiments confirmed the extensive overlap of forebrain- and spinal cord-projecting neurons within the PPNd and the quantitative preponderance of ascending neurons. They also demonstrated that these two projection systems originate largely from separate cell populations since the double-labeled cells always composed less than 5% of the labeled neurons.
The results confirm the existence of a direct PPN projection to the spinal cord. This pathway originates mainly in the PPNd and appears to be quantitatively weaker than the PPN projections to the forebrain. The spinal cord-projecting cells are not spatially segregated from the cells projecting to the basal ganglia, but they represent a separate population of the PPN projection neurons.
The superficial and intermediate gray layers of the superior colliculus are heavily innervated by fibers that utilize the neurotransmitter acetylcholine. The distribution, ultrastructure, and sources of the cholinergic innervation of these layers have been examined in the cat by using a combination of immunocytochemical and axonal transport methods. Putative cholinergic fibers and cells were localized by means of a monoclonal antibody to choline acetyltransferase (ChAT).
ChAT immunoreactive fibers are distributed throughout the depth of the superior colliculus, with particularly dense zones of innervation in the upper part of the superficial grey layer and in the intermediate grey layer. Within the superficial grey layer, the fibers form a continuous, dense band, whereas within the intermediate grey layer the fibers are arranged in clusters or patches. Although the patches are present throughout the rostrocaudal extent of the superior colliculus, they are most prominent in middle to caudal sections.
The structure of the ChAT immunoreactive terminals was examined electron microscopically. The appearance of the terminals is similar in the superficial and intermediate grey layers. They contain closely packed, mostly round vesicles, and form contacts with medium-sized dendrites that exhibit small, but prominent postsynaptic densities; a few of the terminals contact vesicle-containing profiles.
To identify the sources of the cholinergic input to the superior colliculus, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made in the superior colliculus and the sections were processed to demonstrate both the retrograde transport of WGA-HRP and ChAT immunoreactivity. Neurons containing both labels were found in the parabigeminal nucleus, and in the lateral dorsal and pedunculopontine tegmental nuclei of the pontomesencephalic reticular formation. Almost every cell in these nuclei that contained retrograde label was also immunoreactive for ChAT.
The similarities between the laminar distributions of the ChAT terminals and the terminations of the pathway from the parabigeminal nucleus (Graybiel: Brain Res. 145:365–374, '78) support the view that the latter nucleus is a source of the cholinergic fibers in the superficial grey layer. The possibility that the pedunculopontine tegmental nucleus is a source of cholinergic fibers in the deep layers was tested by examining the distribution of labeled fibers following injections of WGA-HRP into this region of the tegmentum. Patches of labeled terminals were found in the intermediate grey layer that resemble in distribution the patches of ChAT immunoreactive fibers in this layer. Only a sparse distribution of labeled terminals was found in the other layers. These results suggest that there are at least two distinct sources of the cholinergic innervation of the superior colliculus: the parabigeminal nucleus for the superficial grey layer and the pedunculopontine tegmental nucleus for the intermediate grey layer. Other potential sources of cholinergic input to the superior colliculus include ChAT immunoreactive neurons that were observed in the present study in the superficial layers of the superior colliculus itself and the cholinergic cells of the lateral dorsal tegmental nucleus that project to the superior colliculus.
Since the pontomesencephalic reticular formation is known to have extensive connections with efferent pathways of the basal ganglia, in a final series of experiments the relationships between the cholinergic pathways from the tegmentum to the superior colliculus and the projections of substantia nigra pars reticulata were explored. Injections of WGA-HRP were madeinto the substantia nigra pars reticulata and alternate sections were processed for either HRP histochemistry or ChAT immunocytochemistry. Within the superior colliculus, the nigrotectal terminals form patches that are approxi-mately equal in number and aligned with the patches of ChAT immunoreac-tive processes. Within the tegmentum, extensive overlap was found between the ChAT immunoreactive cells in the pedunculopontine tegmental nucleus and the terminal field of substantia nigra pars reticulata. These results suggest that there is a close association between the cholinergic innervation of the intermediate grey layer and the nigral outflow of the basal ganglia.
Choline acetyltransferase immunohistochemistry showed that the human rostra1 brainstem contained cholinergic neurons in the oculomotor, trochlear, and parabigeminal nuclei as well as within the reticular formation. The cholinergic neurons of the reticular formation were the most numerous and formed two intersecting constellations. One of these, designated Ch5, reached its peak density within the compact pedunculopontine nucleus but also extended into the regions through which the superior cerebellar peduncle and central tegmental tract course. The second constellation, designated Ch6, was centered around the laterodorsal tegmental nucleus and spread into the central gray and medial longitudinal fasciculus. There was considerable transmitterrelated heterogeneity within the regions containing Ch5 and Ch6. In particular, Ch6 neurons were intermingled with catecholaminergic neurons belonging to the locus coeruleus complex. The lack of confinement within specifiable cytoarchitectonic boundaries and the transmitter heterogeneity justified the transmitter‐specific Ch5 and Ch6 nomenclature for these two groups of cholinergic neurons.
The cholinergic neurons in the nucleus basalis (Ch4) and those of the Ch5‐Ch6 complex were both characterized by perikaryal heteromorphism and isodendritic arborizations. In addition to choline acetyltransferase, the cell bodies in both complexes also had high levels of acetylcholinesterase activity and nonphosphorylated neurofilament protein. However, there were also marked differences in cytochemical signature. For example, the Ch5‐Ch6 neurons had high levels of NADPHd activity, whereas Ch4 neurons did not. On the other hand, the Ch4 neurons had high levels of NGF receptor protein, whereas those of Ch5‐Ch6 did not.
On the basis of animal experiments, it can he assumed that the Ch5 and Ch6 neurons provide the major cholinergic innervation of the human thalamus and that they participate in the neural circuitry of the reticular activating, limbic, and perhaps also extrapyramidal systems.
A total of 260 neurons were recorded in the rostral pontine tegmentum of freely moving cats during the sleep-waking cycle. Of these, 207 neurons (80%) were located in the dorsal pontine tegmentum containing monoaminergic and choline acetyltransferase (ChAT)-immunoreactive, or cholinergic neurons. In addition to presumably monoaminergic PS-off cells (n = 51) showing a cessation of discharge during paradoxical sleep (PS) and presumably cholinergic PGO-on cells (n = 40) exhibiting a burst of discharge just prior to and during ponto-geniculo-occipital (PGO) waves, we observed tonic (n = 108) and phasic (n = 61) neurons exhibiting, respectively, tonic and phasic patterns of discharge during wakefulness and/or paradoxical sleep. Of 87 tonic cells histologically localized in the dorsal pontine tegmentum rich in cholinergic neurons, 46 cells (53%) were identified as giving rise to ascending projections either to the intralaminar thalamic complex (n = 26) or to the ventrolateral posterior hypothalamus (n = 13) or to both (n = 9). Two types of tonic neurons were distinguished: 1) tonic type I neurons (n = 28), showing a tonic pattern and high rates of discharge during both waking and paradoxical sleep as compared with slow wave sleep; and 2) tonic type II neurons (n = 20), exhibiting a tonic pattern of discharge highly specific to the periods of paradoxical sleep. Tonic type I neurons were further divided into two subclasses on the basis of discharge rates during waking: a) rapid (Type I-R; n = 17); and b) slow (Type I-S; n = 11) units with a discharge frequency of more than 12 spikes/s or less than 5 spikes/s, respectively. Like monoaminergic PS-off and cholinergic PGO-on cells, both tonic type II and type I-S cells were characterized by a long spike duration (median: 3.3 and 3.5 ms), as well as by a slow conduction velocity (median = 1.8 and 1.7 m/s). In the light of these data, we discuss the possible cholinergic nature and functional significance of these ascending tonic neurons in the generation of neocortical electroencephalographic desynchronization occurring during waking and paradoxical sleep.
Descending projections from cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei, collectively referred to as the pontomesencephalotegmental (PMT) cholinergic complex, were studied by use of the fluorescent retrograde tracers fluorogold, true blue, or Evans Blue in combination with choline acetyltransferase (ChAT) immunohistochemistry of acetylcholinesterase (AChE) pharmacohistochemistry. Pedunculopontine somata positive for ChAT or staining intensely for AChE were retrogradely labeled with fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12. ChAT-positive cells in both the pedunculopontine and laterodorsal tegmental nuclei demonstrated projections to the vestibular nuclei, the spinal nucleus of the 5th cranial nerve, deep cerebellar nuclei, pontine nuclei, locus ceruleus, raphe magnus nucleus, dorsal raphe nucleus, median raphe nucleus, the medullary reticular nuclei, and the oral and caudal pontine reticular nuclei. Fluorescent tracers used in combination with AChE pharmacohistochemistry corroborated these projections and, in addition, provided evidence for cholinergic pontomesencephalic projections to the lateral reticular nucleus and inferior olive. The majority of retrogradely labeled neurons demonstrating ChAT-like immunoreactivity were found ipsilateral to the injection site, but, in all cases, tracer-containing cholinergic cells contralateral to the infused side of the brain were detected also. More retrogradely labeled cells containing ChAT were observed in the pedunculopontine tegmental than in the laterodorsal tegmental nucleus following tracer injections at all sites with the exceptions of the locus ceruleus and dorsal raphe nucleus where the converse profile was observed. None of the pedunculopontine or laterodorsal tegmental cells immunopositive for ChAT or stained intensely for AChE contained retrogradely transported tracers following dye infusions into the cerebellar cortex or cervical spinal cord. Triple-label experiments using two tracers infused into different sites in the same animal revealed that individual ChAT-immunoreactive cells in the PMT cholinergic complex projected to more than one hindbrain site in some cases and had ascending projections as well. Certain ChAT-positive somata in the pedunculopontine and laterodorsal tegmental nuclei were found in close association with several fiber tracts, including the superior cerebellar peduncle, lateral lemniscus, dorsal tegmental tract, and medial longitudinal fasciculus.
The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN).
Scarce, large (20–30 μm in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat—for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4).
Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH).
Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons.
Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells.
Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons.
On the basis of histochemical and morphological comparison between nigral cholinergic neurons and extranigrally located ChAT-positive cells of the PMT complex, it is concluded that cholinergic neurons in the SN of the rat may be projection neurons of the PMT cholinergic complex ectopically located and that they might play an important role in the regulation of forebrain activation and in locomotion. Our electron microscopic results suggest that substance P and GABA may influence the function of these cells.
The cholinergic innervation of the compact and reticular parts of the substantia nigra in the rat was investigated by use of highly sensitive retrograde and anterograde tract-tracing methods in combination with choline acetyltransferase immunohistochemistry. The fluorescent tracers True Blue, propidium iodide, or fluorogold were infused preferentially into either nigral subnucleus. Cells positive for choline acetyltransferase and retrograde tracer were found in both the pedunculopontine and laterodorsal tegmental nuclei, although considerably more double-labeled somata were observed in the former than in the latter component of the pontomesencephalotegmental cholinergic complex. Approximately 2-3 times more cholinergic cells were labeled in the peduculopontine and laterodorsal tegmental nuclei when tracer injections were centered in the compact nigral subdivision than when infusions of about the same size were confined totally to the reticular part. Infusions of the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin into the pontomesencephalotegmental cholinergic complex resulted in uptake and transport of that label to both nigral subnuclei, and some of the Phaseolus vulgaris leucoagglutinin-accumulating somata and proximal processes also demonstrated choline acetyltransferase-like immunoreactivity. The Phaseolus vulgaris agglutinin-labeled entities in the substantia nigra exhibited terminal-like profiles that were reminiscent of the pattern of nigral choline acetyltransferase-positive puncta demonstrated immunohistochemically by use of nickel ammonium sulfate enhancement of the final reaction product in the avidin-biotin procedure. These observations strongly support the contention that the pontomesencephalotegmental cholinergic complex is the major source of cholinergic projections to both the compact and reticular portions of the rat substantia nigra.
The electrophysiological characteristics of neurons of the nucleus tegmenti pedunculopontinus (PPN), in particular of those projecting to the substantia nigra (SN), and the reciprocal influence between the PPN and SN were investigated in normal and decorticated rats. In intact animals 65 of the 363 PPN recorded neurons (17.9%) were activated antidromically by SN stimulation, 96 (26.3%) were inhibited after stimulation while 43 (11.8%) were activated. In decorticated rats excitatory responses were decreased (4.8%) while antidromic and inhibitory responses did not change substantially. Electrical stimulation of the PPN induced a brief short-latency excitation of SN neurons (26/77, 33.7%) which was not modified by removing the cortex bilaterally 7-10 days prior to the recording session. This excluded the possibility that corticofugal fibers could be involved in the excitatory responses evoked by PPN stimulation in SN neurons. The latency of the antidromic response evoked in PPN cells by SN stimulation ranged from 0.5 to 12.0 ms and the estimated conduction velocity of these PPN output neurons ranged from 1.1 to less than 0.5 m/s. The electrophysiological heterogeneity of PPN cells was supported also by the fact that two types of neurons, both projecting to the SN, could be distinguished on the basis of their spontaneous firing rate and impulse waveform. The first had a low spontaneous activity (0.5-8 spikes/s) with a triphasic impulse which lasted 3-4 ms. The second had a high firing rate (15-20 spikes/s) and its impulse was usually biphasic and not longer than 3 ms.(ABSTRACT TRUNCATED AT 250 WORDS)
The nature of the synaptic transmitter involved in the excitatory fibers linking the nucleus tegmenti pedunculopontinus (PPN) to the pars compacta of the substantia nigra (SNPC) was investigated using microiontophoretic techniques in rats anesthetized with ketamine. Among the SNPC cells activated orthodromically by PPN electrical stimulation, only a few cells were weakly excited by iontophoretically administered acetylcholine (Ach) while most were not affected. Conversely all cells were promptly and powerfully excited by short pulses of glutamate (GLU). The administration of the GLU antagonists glutamic acid diethyl ester (GDEE) and D-alpha-aminoadipic acid (DAA) reversibly and simultaneously suppressed both the PPN-evoked orthodromic response and the GLU-induced excitation of SNPC cells without affecting their response to iontophoretic Ach. GDEE was more effective than DAA in counteracting the synaptically evoked excitation. On the other hand, atropine, while antagonizing the Ach response in those cells which were cholinoceptive, did not affect either the PPN-evoked or the GLU-induced excitation. Hence, despite the presence of cholinergic cells in the PPN region, Ach does not appear to be involved in the excitatory PPN-SNPC pathway. The present findings suggest that the excitatory PPN fibers innervating the SNPC may utilize GLU or a closely related amino acid as a neurotransmitter.
The pedunculopontine tegmental nucleus (PPTn) was originally defined on cytoarchitectonic grounds in humans. We have employed cytoarchitectonic, cytochemical, and connectional criteria to define a homologous cell group in the rat. A detailed cytoarchitectonic delineation of the mesopontine tegmentum, including the PPTn, was performed employing tissue stained for Nissl substance. Choline acetyltransferase (ChAT) immunostained tissue was then analyzed in order to investigate the relationship of cholinergic perikarya, dendritic arborizations, and axonal trajectories within this cytoarchitectonic scheme. To confirm some of our cytoarchitectonic delineations, the relationships between neuronal elements staining for ChAT and tyrosine hydroxylase were investigated on tissue stained immunohistochemically for the simultaneous demonstration of these two enzymes.
The PPTn consists of large, multipolar neurons, all of which stain immunohistochemically for ChAT. It is present within cross‐sections that also include the A‐6 through A‐9 catecholamine cell groups and is traversed by catecholaminergic axons within the dorsal tegmental bundle and central tegmental tract. The dendrites of PPTn neurons respect several nuclear boundaries and are oriented perpendicularly to several well‐defined fiber tracts. Cholinergic axons ascend from the mesopontine tegmentum through the dorsal tegmental bundle and a more lateral dorsal ascending pathway. A portion of the latter terminates within the lateral geniculate nucleus.
It has been widely believed that the PPTn is reciprocally connected with several extrapyramidal structures, including the globus pallidus and substantia nigra pars reticulata. Therefore, the relationships of pallidotegmental and nigrotegmental pathways to the PPTn were investigated employing the anterograde autoradiographic methodology. The reciprocity of tegmental connections with the substantia nigra and entopeduncular nucleus was investigated employing combined WGA‐HRP injections and ChAT immunohistochemistry.
The pallido‐ and nigrotegmental terminal fields did not coincide with the PPTn, but, rather, were located just medial and dorsomedial to it (the midbrain extrapyramidal area). The midbrain extrapyramidal area, but not the PPTn, was reciprocally connected with the substantia nigra and entope‐duncular nucleus. We discuss these results in light of other cytoarchitec‐tonic, cytochemical, connectional, and physiologic studies of the functional anatomy of the mesopontine tegmentum.
This study demonstrates the pedunculopontine tegmental nucleus (PPN) to be the source of a major cholinergic projection to the rat substantia nigra (SN). Neurons of the PPN were double-labeled utilizing choline acetyltransferase immunocytochemistry combined with retrograde transport of horseradish peroxidase (HRP). The cholinergic projection to the SN originates from neurons located in predominately the ipsilateral and rostral portions of the PPN. Other cholinergic neurons that were also retrogradely labeled with HRP were located in the caudal PPN and in the lateral dorsal tegmental nucleus. A non-cholinergic projection from this region was also identified. These findings indicate that the PPN may be topographically organized with respect to its efferent projections. In addition, this study provides evidence for an extrinsic source of acetylcholine to the basal ganglia and implicates the PPN as a source of potentially significant influence over basal ganglia function.
Biocytin is a biotin-lysine complex of low molecular weight containing about 65% biotin, which retains a high affinity for avidin. Since the latter molecule has been conjugated to several histochemical markers, the use of biocytin as an intracellular marker was investigated. Electrodes were filled with a solution of 4-6% biocytin dissolved in 0.5 M KCl and 0.05 M Tris buffer, pH 7-7.6. Neurons were recorded intracellularly in the supraoptic nucleus of an explant preparation of the rat supraoptico-neurohypophysial system and injected for 1-20 min with either hyperpolarizing or depolarizing current. Following variable recovery times, the explants were fixed in either 10% formalin or 4% paraformaldehyde overnight, sectioned on a vibratome, and incubated with the avidin-biotin complex (ABC) or avidin which had been conjugated to fluorescein, rhodamine, Texas Red or horseradish peroxidase and containing 1% Triton-X 100. A high percentage of injected neurons were recovered using each of the labels with about equal success. Both negative or positive current injection could be used with little electrode clogging. Labeling with fluorescent conjugates was qualitatively similar to that of Lucifer Yellow, whereas labeling with avidin coupled to horseradish peroxidase or with ABC was qualitatively similar to filling neurons directly with horseradish peroxidase. The advantages of this technique are the ease of injection of biocytin and the versatility in allowing the investigator to choose among light-emitting and light-absorbing images.
Putative cholinergic axons and synaptic endings were demonstrated in the substantia nigra (SN) of the rat by light and electron microscopy on the basis of the localization of choline acetyltransferase (ChAT) immunoreactivity. The distribution of ChAT immunoreactivity in the SN as demonstrated by light microscopy revealed a modest network of ChAT-immunoreactive beaded axons in the SNc, in comparison to a relatively sparse distribution in the SNr. These axonal profiles were most dense in the middle of the rostral-caudal extent of the SNc and appeared to be concentrated in the middle third of the medial-lateral extent. By electron microscopy, unmyelinated, small diameter (0.25 micron) ChAT-immuno-reactive axons were observed interspersed among numerous other non-immunoreactive axons in the SNc. ChAT-immunoreactive synaptic endings were observed in juxtaposition to small caliber (0.5 micron) non-immunoreactive dendrites, and contained numerous spheroidal synaptic vesicles and occasional mitochondria. Synaptic contact zones were characterized by an accumulation of synaptic vesicles along the presynaptic membrane, and a prominent postsynaptic densification producing an asymmetrical pre-/postsynaptic membrane profile typical of excitatory synapses. These findings provide direct evidence for a cholinergic innervation of the SN, and suggest that this input may have an excitatory effect on neuronal elements in the SNc.
Recordings of single unit activity in the posterior midbrain of the cat were carried out in the "fictive spontaneous locomotion" preparation. Neuronal activity was studied in relation to the onset, alternation and termination of cyclic hindlimb neurographic activity in the precollicular-postmammillary transected animal. Histochemical identification of pedunculopontine (nicotinamide adenine dinuceotide phosphate-diaphorase positive) neurons allowed the localization of recording sites in relation to this nucleus. Neurons located in the area of the cuneiform nucleus dorsal to the pedunculopontine nucleus were found to be related preferentially to cyclic (bursting) neurographic activity, while neurons in the area of the pedunculopontine were found to be related preferentially to the onset ("on") or termination ("off") of cycling episodes. Different populations of cells in the area appeared to be related to the frequency of alternation (bursting) compared with the duration of the cyclic episodes (on/off). While the area of the cuneiform-pedunculopontine nucleus has been found to be equivalent to the mesencephalic locomotor region, the same area has been found to be related to other rhythmic activities (e.g. respiratory, masticatory, sleep cycle, pressor, vesico-motor, etc.). A hypothesis is proposed to account for the weight of evidence implicating the same region in a host of distinct rhythmic activities. This hypothesis suggest that an oscillatory reverberation between cholinergic (pedunculopontine, laterodorsal tegmental nuclei) and aminergic (locus coeruleus, substantia nigra) centers is responsible for generating the various function-related "frequencies" (bursting) or "states" (on/off) of activity.
This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.
Electrolytic lesion was placed in the nucleus tegmenti pedunculopontinus pars compacta (TPC) in cats, which were injected simultaneously with horseradish peroxidase (HRP) into the caudate nucleus (Cd). After a survival period of 4 days, the substantia nigra pars compacta (SNC) was examined electron microscopically. Degenerating axon terminals were found to make asymmetric synaptic contacts on dendrites of SNC neurons which were retrogradely labeled with HRP. Thus, TPC neurons were indicated to project monosynaptically to SNC neurons which sent their axons to the Cd.
The coexistence of immunoreactivities for choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in some brain regions of the rat, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for ChAT and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the retina, cerebral cortex, basal forebrain and spinal cord, colocalization of ChAT-like and GAD-like or GABA-like immunoreactivities was observed in some cell types, whereas no such colocalization was observed in cells in the striatum or brainstem. In the retina, the majority of ChAT-like immunoreactive (ChAT-LI) amacrine cells contained GABA-like or GAD-like immunoreactivity. About half of the ChAT-LI neurons in the cerebral cortex showed GABA-like immunoreactivity. In the basal forebrain only a small proportion of ChAT-LI neurons (0.6%) contained GAD-like immunoreactivity. In the spinal cord, about one-third of ChAT-LI central canal cluster cells and about half of ChAT-LI dorsal horn cells showed GAD-like and/or GABA-like immunoreactivities. These observations indicate the possible coexistence of two classical transmitters, GABA and acetylcholine, in various brain regions and spinal cord of the rat.
The present study provides evidence of a synaptic contact between terminals of pedunculopontine neurons and cells of substantia nigra pars compacta (SNPC) in the rat. Three types of projective neurons observed in Golgi stained preparations of the pedunculopontine region might be the source of this afferent pathway to the substantia nigra. These PPN projective neurons were small, medium and large sized cells. After placing a small electrolytic lesion into the pedunculopontine area two types of degenerated terminals were found in the SNPC, a small and a larger one. These degenerated terminals contained round synaptic vesicles, thus suggesting that the pedunculopontine input to the SNPC is monosynaptic and excitatory in nature.
We combined the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry to study the projections of cholinergic and non-cholinergic neurons of the upper brainstem core to rostral and caudal intralaminar thalamic nuclei, reticular thalamic complex and zona incerta in the cat. After wheat germ agglutinin horseradish peroxidase injections in the rostral pole of the reticular thalamic nucleus, the distribution and amount of retrogradely labeled brainstem neurons were similar to those found after tracer injection in thalamic relay nuclei (see preceding paper⁴²). After wheat germ agglutinin horseradish peroxidase injections in the caudal intralaminar centrum medianum parafascicular complex, rostral intralaminar central lateral paracentral wing, and zona incerta, the numbers of retrogradely labeled brainstem neurons were more than three times higher than those found after injections in thalamic relay nuclei. The larger numbers of horseradish peroxidase-positive brainstem reticular neurons after tracer injections in intralaminar or zona incerta injections results from a more substantial proportion of labeled neurons in the central tegmental field at rostral midbrain (perirubral) levels and in the ventromedial part of the pontine reticular formation, ipsi- and contralaterally to the injection site. Of all retrogradely labeled neurons in the caudal midbrain core at the level of the cholinergic peribrachial area and laterodorsal tegmental nucleus. 45–50% were also choline acetyltransferase-positive alter the injections into central lateral paracentral and reticular nuclei, while only 25% were also choline acetyltransferase-positive after the injection into the centrum medianum parafascicular complex.
The projections of brainstem core neurons to relay and associational thalamic nuclei were studied in the cat and macaque monkey by combining the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry. All major sensory (medial geniculate, lateral geniculate, ventrobasal), motor (ventroanterior, ventrolateral, ventromedial), associational (mediodorsal, pulvinar, lateral posterior) and limbic (anteromedial, anteroventral) thalamic nuclei of the cat were found to receive projections from cholinergic neurons located in the peribrachial area of the pedunculopontine nucleus and in the laterodorsal tegmental nucleus as well as from non-cholinergic neurons in the rostral (perirubral) part of the central tegmental mesencephalic field. Specific relay nuclei receive less than 10% of their brainstem afferents from non-cholinergic neurons located at rostral midbrain levels and receive 85-96% of their brainstem innervation from a region at midbrain-pontine junction where the cholinergic peribrachial area and laterodorsal tegmental nucleus are maximally developed. Of the total number of horseradish peroxidase-positive brainstem neurons seen after injections in various specific relay nuclei, the double-labeled (horseradish peroxidase + choline acetyltransferase) neurons represent approximately 70-85%. Three to eight times more numerous horseradish peroxidase-labeled brainstem cells were found after injections in associational (mediodorsal and pulvinar-lateral posterior complex) and diffusely cortically-projecting (ventromedial) thalamic nuclei of cat than after injections in specific relay nuclei. The striking retrograde cell labeling observed after injections in nuclei with associative functions and widespread cortical projections was due to massive afferentation from non-cholinergic parts of the midbrain and pontine reticular formation, on both ipsi- and contralateral sides. After wheat germ agglutinin-horseradish peroxidase injections in the associative pulvinar-lateral posterior complex and mediodorsal nucleus of Macaca sylvana, 45-50% of horseradish peroxidase-positive brainstem peribrachial neurons were also choline acetyltransferase-positive. While cells in the medial part of the cholinergic peribrachial area were found to project especially towards the pulvinar-lateral posterior nuclear complex in monkey, the retrograde cell labeling seen after the mediodorsal injection was mostly confined to the lateral part of both dorsal and ventral aspects of the peribrachial area.(ABSTRACT TRUNCATED AT 400 WORDS)
Dopaminergic neurons of the substantia nigra pars compacta are excited by nicotine and acetylcholine, and possess both high-affinity nicotine binding sites and intense acetylcholinesterase activity, consistent with a cholinoceptive role. A probable source of cholinergic afferents is the pedunculopontine nucleus, which forms part of a prominent group of cholinergic perikarya located caudal to the substantia nigra in the tegmentum. Although pedunculopontine efferents, many of them cholinergic, project to the substantia nigra pars compacta, it has not been established whether they terminate in this structure. In the first experiment, which combined retrograde tracing with immunohistochemical visualization of cholinergic neurons, cholinergic cells in and around the pedunculopontine nucleus were found to send projections to the substantia nigra. This projection was almost completely ipsilateral. Subsequent experiments employed anaesthetized rats; kainate was microinfused into tegmental sites in order to stimulate local cholinergic perikarya, and concurrently, extracellular recordings were made of single dopaminergic neurons in the substantia nigra. Consistent with our anatomical findings, unilateral microinfusion of kainic acid in or near the pedunculopontine nucleus increased the firing rate of dopaminergic neurons situated remotely in the ipsilateral substantia nigra. The kainate-induced excitation of nigral dopaminergic neurons was dose-related and was prevented by intravenous administration of the centrally-acting nicotinic cholinergic antagonist mecamylamine. These results suggest that cholinergic perikarya in the vicinity of the pedunculopontine tegmental nucleus innervate dopaminergic neurons in the substantia nigra pars compacta via nicotinic receptors.
Acetylcholinesterase (AChE) has been localized by histochemistry in the superior colliculus and in the tegmentum of the caudal midbrain and rostral pons of the rat. The pattern of AChE localization in the superior colliculus was characterized by homogeneous staining in the superficial layers and patchlike staining in the intermediate gray layer. In the tegmentum, AChE was localized in the pedunculopontine nucleus (PPN), beginning rostrally at the caudal pole of the substantia nigra and extending caudally to the level of the parabrachial nuclei, and in the lateral dorsal tegmental nucleus (LDTN) of the central gray. The localization of AChE in these nuclei overlapped the distribution of neurons stained by immunohistochemistry using an antibody to choline acetyltransferase (CHAT), the synthesizing enzyme of the neurotransmitter acetylcholine. Other neighboring areas that were stained with AChE, but that did not contain ChAT‐immunoreactive neurons, included the microcellular tegmental nucleus and the ventral tegmental nucleus.
Neurons in the PPN and LDTN were determined to be potential sources of the cholinergic projection to the intermediate gray layer of the rat superior colliculus by double labelling with retrograde transport of horseradish peroxidase (HRP) combined with the immunohistochemical localization of ChAT. Three populations of neurons were identified. A predominantly ipsilateral ChAT‐immunoreactive population was located in the pars compacta subdivision of PPN (PPNpc). Retrograde HRP‐labelled neurons in the pars dissipata subdivison of the PPN (PPNpd), located ventral to the superior cerebellar peduncle (SCP) at the level of the inferior colliculus, composed a second population that was predominantly contralateral but was not ChAT immunoreactive. A third population of retrogradely labelled neurons was predominantly ipsilateral and ChAT immunoreactive and was located in the LDTN. These findings compared favorably with the full extent of the projection from this tegmental region revealed by retrograde transport of HRP from the superior colliculus when more compatible fixation and chromogen procedures were used.
The results suggest that the PPN and the LDTN are two sources of the cholinergic input to the superior colliculus. Since the PPN also has extensive efferent, and afferent, connections with basal‐ganglia‐related structures, this cholinergic excitatory input to the superior colliculus, like the GABAergic inhibitory input from the substantia nigra pars reticulata, may provide the basis for an additional influence of the basal ganglia on visuomotor behavior.
The organization of nucleus tegmenti pedunculopontinus (PPN) projections to the basal ganglia and thalamus was studied in the rat by using retrograde transport of fluorescent dyes. Fast blue was injected into the substantia nigra (SN) while Nuclear yellow was delivered to one of the following nuclei: globus pallidus (GP), entopeduncular nucleus, subthalamic nucleus (STN) or parafascicular nucleus of the thalamus. Retrogradely labeled cells were observed throughout the PPN without topographical arrangement. The cells labeled from the SN outnumbered those labeled from other structures. In all cases the majority of cells were single labeled and only a few cells double labeled from SN-GP or SN-STN were found. Labeled cells were either fusiform or multipolar in shape. These data suggest that distinct PPN cells project to their basal ganglia and thalamic targets without a prominent branched organization.
The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.